Sample records for single biological sample
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9 CFR 113.3 - Sampling of biological products.
Code of Federal Regulations, 2013 CFR
2013-01-01
... bacterial vaccines; (iii) Two samples of Coccidiosis Vaccine; (iv) Eighteen samples of Rabies Vaccine...) Twenty-two single-dose or 14 multiple-dose samples of Rabies Vaccine, Killed Virus; (viii) Sixteen single...
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9 CFR 113.3 - Sampling of biological products.
Code of Federal Regulations, 2014 CFR
2014-01-01
... bacterial vaccines; (iii) Two samples of Coccidiosis Vaccine; (iv) Eighteen samples of Rabies Vaccine...) Twenty-two single-dose or 14 multiple-dose samples of Rabies Vaccine, Killed Virus; (viii) Sixteen single...
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9 CFR 113.3 - Sampling of biological products.
Code of Federal Regulations, 2012 CFR
2012-01-01
... bacterial vaccines; (iii) Two samples of Coccidiosis Vaccine; (iv) Eighteen samples of Rabies Vaccine...) Twenty-two single-dose or 14 multiple-dose samples of Rabies Vaccine, Killed Virus; (viii) Sixteen single...
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Plant Systems Biology at the Single-Cell Level.
Libault, Marc; Pingault, Lise; Zogli, Prince; Schiefelbein, John
2017-11-01
Our understanding of plant biology is increasingly being built upon studies using 'omics and system biology approaches performed at the level of the entire plant, organ, or tissue. Although these approaches open new avenues to better understand plant biology, they suffer from the cellular complexity of the analyzed sample. Recent methodological advances now allow plant scientists to overcome this limitation and enable biological analyses of single-cells or single-cell-types. Coupled with the development of bioinformatics and functional genomics resources, these studies provide opportunities for high-resolution systems analyses of plant phenomena. In this review, we describe the recent advances, current challenges, and future directions in exploring the biology of single-cells and single-cell-types to enhance our understanding of plant biology as a system. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Martinez, Jennifer S [Santa Fe, NM; Swanson, Basil I [Los Alamos, NM; Grace, Karen M [Los Alamos, NM; Grace, Wynne K [Los Alamos, NM; Shreve, Andrew P [Santa Fe, NM
2009-06-02
An assay element is described including recognition ligands bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of a biological target is described including injecting a biological target-containing sample into a sensor cell including the assay element, with the recognition ligands adapted for binding to selected biological targets, maintaining the sample within the sensor cell for time sufficient for binding to occur between selected biological targets within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting the fluorescent-label in any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.
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Vibrational spectroscopy for imaging single microbial cells in complex biological samples
Harrison, Jesse P.; Berry, David
2017-04-13
Here, vibrational spectroscopy is increasingly used for the rapid and non-destructive imaging of environmental and medical samples. Both Raman and Fourier-transform infrared (FT-IR) imaging have been applied to obtain detailed information on the chemical composition of biological materials, ranging from single microbial cells to tissues. Due to its compatibility with methods such as stable isotope labeling for the monitoring of cellular activities, vibrational spectroscopy also holds considerable power as a tool in microbial ecology. Chemical imaging of undisturbed biological systems (such as live cells in their native habitats) presents unique challenges due to the physical and chemical complexity of themore » samples, potential for spectral interference, and frequent need for real-time measurements. This Mini Review provides a critical synthesis of recent applications of Raman and FT-IR spectroscopy for characterizing complex biological samples, with a focus on developments in single-cell imaging. We also discuss how new spectroscopic methods could be used to overcome current limitations of singlecell analyses. Given the inherent complementarity of Raman and FT-IR spectroscopic methods, we discuss how combining these approaches could enable us to obtain new insights into biological activities either in situ or under conditions that simulate selected properties of the natural environment.« less
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Vibrational spectroscopy for imaging single microbial cells in complex biological samples
DOE Office of Scientific and Technical Information (OSTI.GOV)
Harrison, Jesse P.; Berry, David
Here, vibrational spectroscopy is increasingly used for the rapid and non-destructive imaging of environmental and medical samples. Both Raman and Fourier-transform infrared (FT-IR) imaging have been applied to obtain detailed information on the chemical composition of biological materials, ranging from single microbial cells to tissues. Due to its compatibility with methods such as stable isotope labeling for the monitoring of cellular activities, vibrational spectroscopy also holds considerable power as a tool in microbial ecology. Chemical imaging of undisturbed biological systems (such as live cells in their native habitats) presents unique challenges due to the physical and chemical complexity of themore » samples, potential for spectral interference, and frequent need for real-time measurements. This Mini Review provides a critical synthesis of recent applications of Raman and FT-IR spectroscopy for characterizing complex biological samples, with a focus on developments in single-cell imaging. We also discuss how new spectroscopic methods could be used to overcome current limitations of singlecell analyses. Given the inherent complementarity of Raman and FT-IR spectroscopic methods, we discuss how combining these approaches could enable us to obtain new insights into biological activities either in situ or under conditions that simulate selected properties of the natural environment.« less
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Single-molecule fluorescence microscopy review: shedding new light on old problems
Shashkova, Sviatlana
2017-01-01
Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. PMID:28694303
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Cuozzo, Frank P; Rasoazanabary, Emilienne; Godfrey, Laurie R; Sauther, Michelle L; Youssouf, Ibrahim Antho; LaFleur, Marni M
2013-01-01
A thorough knowledge of biological variation in extant primates is imperative for interpreting variation, and for delineating species in primate biology and paleobiology. This is especially the case given the recent, rapid taxonomic expansion in many primate groups, notably among small-bodied nocturnal forms. Here we present data on dental, cranial, and pelage variation in a single-locality museum sample of mouse lemurs from Amboasary, Madagascar. To interpret these data, we include comparative information from other museum samples, and from a newly collected mouse lemur skeletal sample from the Beza Mahafaly Special Reserve (BMSR), Madagascar. We scored forty dental traits (n = 126) and three pelage variants (n = 19), and collected 21 cranial/dental measures. Most dental traits exhibit variable frequencies, with some only rarely present. Individual dental variants include misshapen and supernumerary teeth. All Amboasary pelage specimens display a "reversed V" on the cap, and a distinct dorsal median stripe on the back. All but two displayed the dominant gray-brown pelage coloration typical of Microcebus griseorufus. Cranial and dental metric variability are each quite low, and craniometric variation does not illustrate heteroscedasticity. To assess whether this sample represents a single species, we compared dental and pelage variation to a documented, single-species M. griseorufus sample from BMSR. As at Amboasary, BMSR mouse lemurs display limited odontometric variation and wide variation in non-metric dental traits. In contrast, BMSR mouse lemurs display diverse pelage, despite reported genetic homogeneity. Ranges of dental and pelage variation at BMSR and Amboasary overlap. Thus, we conclude that the Amboasary mouse lemurs represent a single species - most likely (in the absence of genetic data to the contrary) M. griseorufus, and we reject their previous allocation to Microcebus murinus. Patterns of variation in the Amboasary sample provide a comparative template for recognizing the degree of variation manifested in a single primate population, and by implication, they provide minimum values for this species' intraspecific variation. Finally, discordance between different biological systems in our mouse lemur samples illustrates the need to examine multiple systems when conducting taxonomic analyses among living or fossil primates. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Ferrara, Toni L; Boughton, Philip; Slavich, Eve; Wroe, Stephen
2013-01-01
Nanomechanical testing methods that are suitable for a range of hydrated tissues are crucial for understanding biological systems. Nanoindentation of tissues can provide valuable insights into biology, tissue engineering and biomimetic design. However, testing hydrated biological samples still remains a significant challenge. Shark jaw cartilage is an ideal substrate for developing a method to test hydrated tissues because it is a unique heterogeneous composite of both mineralized (hard) and non-mineralized (soft) layers and possesses a jaw geometry that is challenging to test mechanically. The aim of this study is to develop a novel method for obtaining multidirectional nanomechanical properties for both layers of jaw cartilage from a single sample, taken from the great white shark (Carcharodon carcharias). A method for obtaining multidirectional data from a single sample is necessary for examining tissue mechanics in this shark because it is a protected species and hence samples may be difficult to obtain. Results show that this method maintains hydration of samples that would otherwise rapidly dehydrate. Our study is the first analysis of nanomechanical properties of great white shark jaw cartilage. Variation in nanomechanical properties were detected in different orthogonal directions for both layers of jaw cartilage in this species. The data further suggest that the mineralized layer of shark jaw cartilage is less stiff than previously posited. Our method allows multidirectional nanomechanical properties to be obtained from a single, small, hydrated heterogeneous sample. Our technique is therefore suitable for use when specimens are rare, valuable or limited in quantity, such as samples obtained from endangered species or pathological tissues. We also outline a method for tip-to-optic calibration that facilitates nanoindentation of soft biological tissues. Our technique may help address the critical need for a nanomechanical testing method that is applicable to a variety of hydrated biological materials whether soft or hard.
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NASA Astrophysics Data System (ADS)
Al-Mashat, H.; Kristensen, L.; Sultana, C. M.; Prather, K. A.
2016-12-01
The ability to distinguish types of particles present within a cloud is important for determining accurate inputs to climate models. The chemical composition of particles within cloud liquid droplets and ice crystals can have a significant impact on the timing, location, and amount of precipitation that falls. Precipitation efficiency is increased by the presence of ice crystals in clouds, and both mineral dust and biological aerosols have been shown to be effective ice nucleating particles (INPs) in the atmosphere. A current challenge in aerosol science is distinguishing mineral dust and biological material in the analysis of real-time, ambient, single-particle mass spectral data. Single-particle mass spectrometers are capable of measuring the size-resolved chemical composition of individual atmospheric particles. However, there is no consistent analytical method for distinguishing dust and biological aerosols. Sampling and characterization of control samples (i.e. of known identity) of mineral dust and bacteria were performed by the Aerosol Time-of-Flight Mass Spectrometer (ATOFMS) as part of the Fifth Ice Nucleation (FIN01) Workshop at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) facility in Karlsruhe, Germany. Using data collected by the ATOFMS of control samples, a new metric has been developed to classify single particles as dust or biological independent of spectral cluster analysis. This method, involving the use of a ratio of mass spectral peak areas for organic nitrogen and silicates, is easily reproducible and does not rely on extensive knowledge of particle chemistry or the ionization characteristics of mass spectrometers. This represents a step toward rapidly distinguishing particle types responsible for ice nucleation activity during real-time sampling in clouds. The ability to distinguish types of particles present within a cloud is important for determining accurate inputs to climate models. The chemical composition of particles within cloud liquid droplets and ice crystals can have a significant impact on the timing, location, and amount of precipitation that falls. Precipitation efficiency is increased by the presence of ice crystals in clouds, and both mineral dust and biological aerosols have been shown to be effective ice nucleating particles (INPs) in the atmosphere. A current challenge in aerosol science is distinguishing mineral dust and biological material in the analysis of real-time, ambient, single-particle mass spectral data. Single-particle mass spectrometers are capable of measuring the size-resolved chemical composition of individual atmospheric particles. However, there is no consistent analytical method for distinguishing dust and biological aerosols. Sampling and characterization of control samples (i.e. of known identity) of mineral dust and bacteria were performed by the Aerosol Time-of-Flight Mass Spectrometer (ATOFMS) as part of the Fifth Ice Nucleation (FIN01) Workshop at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) facility in Karlsruhe, Germany. Using data collected by the ATOFMS of control samples, a new metric has been developed to classify single particles as dust or biological independent of spectral cluster analysis. This method, involving the use of a ratio of mass spectral peak areas for organic nitrogen and silicates, is easily reproducible and does not rely on extensive knowledge of particle chemistry or the ionization characteristics of mass spectrometers. This represents a step toward rapidly distinguishing particle types responsible for ice nucleation activity during real-time sampling in clouds.
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Comparability among four invertebrate sampling methods, Fountain Creek Basin, Colorado, 2010-2012
Zuellig, Robert E.; Bruce, James F.; Stogner, Sr., Robert W.; Brown, Krystal D.
2014-01-01
The U.S. Geological Survey, in cooperation with Colorado Springs City Engineering and Colorado Springs Utilities, designed a study to determine if sampling method and sample timing resulted in comparable samples and assessments of biological condition. To accomplish this task, annual invertebrate samples were collected concurrently using four sampling methods at 15 U.S. Geological Survey streamflow gages in the Fountain Creek basin from 2010 to 2012. Collectively, the four methods are used by local (U.S. Geological Survey cooperative monitoring program) and State monitoring programs (Colorado Department of Public Health and Environment) in the Fountain Creek basin to produce two distinct sample types for each program that target single-and multiple-habitats. This study found distinguishable differences between single-and multi-habitat sample types using both community similarities and multi-metric index values, while methods from each program within sample type were comparable. This indicates that the Colorado Department of Public Health and Environment methods were compatible with the cooperative monitoring program methods within multi-and single-habitat sample types. Comparisons between September and October samples found distinguishable differences based on community similarities for both sample types, whereas only differences were found for single-habitat samples when multi-metric index values were considered. At one site, differences between September and October index values from single-habitat samples resulted in opposing assessments of biological condition. Direct application of the results to inform the revision of the existing Fountain Creek basin U.S. Geological Survey cooperative monitoring program are discussed.
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Massively parallel nanowell-based single-cell gene expression profiling.
Goldstein, Leonard D; Chen, Ying-Jiun Jasmine; Dunne, Jude; Mir, Alain; Hubschle, Hermann; Guillory, Joseph; Yuan, Wenlin; Zhang, Jingli; Stinson, Jeremy; Jaiswal, Bijay; Pahuja, Kanika Bajaj; Mann, Ishminder; Schaal, Thomas; Chan, Leo; Anandakrishnan, Sangeetha; Lin, Chun-Wah; Espinoza, Patricio; Husain, Syed; Shapiro, Harris; Swaminathan, Karthikeyan; Wei, Sherry; Srinivasan, Maithreyan; Seshagiri, Somasekar; Modrusan, Zora
2017-07-07
Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.
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Non-invasive optoacoustic probing of the density and stiffness of single biological cells
NASA Astrophysics Data System (ADS)
Dehoux, T.; Audoin, B.
2012-12-01
Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accurately for a reference fluid. Similar analysis performed in a single vegetal cell also suggests high sensitivity to the state of the transducer-cell interface, and notably to the presence of the plasma membrane that encloses the cell vacuole.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Merolle, L., E-mail: lucia.merolle@elettra.eu; Gianoncelli, A.; Malucelli, E., E-mail: emil.malucelli@unibo.it
2016-01-28
Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the majormore » obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.« less
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Monitoring biological aerosols using UV fluorescence
NASA Astrophysics Data System (ADS)
Eversole, Jay D.; Roselle, Dominick; Seaver, Mark E.
1999-01-01
An apparatus has been designed and constructed to continuously monitor the number density, size, and fluorescent emission of ambient aerosol particles. The application of fluorescence to biological particles suspended in the atmosphere requires laser excitation in the UV spectral region. In this study, a Nd:YAG laser is quadrupled to provide a 266 nm wavelength to excite emission from single micrometer-sized particles in air. Fluorescent emission is used to continuously identify aerosol particles of biological origin. For calibration, biological samples of Bacillus subtilis spores and vegetative cells, Esherichia coli, Bacillus thuringiensis and Erwinia herbicola vegetative cells were prepared as suspensions in water and nebulized to produce aerosols. Detection of single aerosol particles, provides elastic scattering response as well as fluorescent emission in two spectral bands simultaneously. Our efforts have focuses on empirical characterization of the emission and scattering characteristics of various bacterial samples to determine the feasibility of optical discrimination between different cell types. Preliminary spectroscopic evidence suggest that different samples can be distinguished as separate bio-aerosol groups. In addition to controlled sample results, we will also discuss the most recent result on the effectiveness of detection outdoor releases and variations in environmental backgrounds.
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Jiao, Shuliang; Todorović, Milos; Stoica, George; Wang, Lihong V
2005-09-10
We report on a new configuration of fiber-based polarization-sensitive Mueller matrix optical coherence tomography that permits the acquisition of the round-trip Jones matrix of a biological sample using only one light source and a single depth scan. In this new configuration, a polarization modulator is used in the source arm to continuously modulate the incident polarization state for both the reference and the sample arms. The Jones matrix of the sample can be calculated from the two frequency terms in the two detection channels. The first term is modulated by the carrier frequency, which is determined by the longitudinal scanning mechanism, whereas the other term is modulated by the beat frequency between the carrier frequency and the second harmonic of the modulation frequency of the polarization modulator. One important feature of this system is that, for the first time to our knowledge, the Jones matrix of the sample can be calculated with a single detection channel and a single measurement when diattenuation is negligible. The system was successfully tested by imaging both standard polarization elements and biological samples.
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Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming
2017-03-07
The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.
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Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming
2017-01-01
The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules. PMID:28223513
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Reinhold, William C
2015-12-10
There is currently a split within the cancer research community between traditional molecular biological hypothesis-driven and the more recent "omic" forms or research. While the molecular biological approach employs the tried and true single alteration-single response formulations of experimentation, the omic employs broad-based assay or sample collection approaches that generate large volumes of data. How to integrate the benefits of these two approaches in an efficient and productive fashion remains an outstanding issue. Ideally, one would merge the understandability, exactness, simplicity, and testability of the molecular biological approach, with the larger amounts of data, simultaneous consideration of multiple alterations, consideration of genes both of known interest along with the novel, cross-sample comparisons among cell lines and patient samples, and consideration of directed questions while simultaneously gaining exposure to the novel provided by the omic approach. While at the current time integration of the two disciplines remains problematic, attempts to do so are ongoing, and will be necessary for the understanding of the large cell line screens including the Developmental Therapeutics Program's NCI-60, the Broad Institute's Cancer Cell Line Encyclopedia, and the Wellcome Trust Sanger Institute's Cancer Genome Project, as well as the the Cancer Genome Atlas clinical samples project. Going forward there is significant benefit to be had from the integration of the molecular biological and the omic forms or research, with the desired goal being improved translational understanding and application.
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Chen, S.; Deng, J.; Nashed, Y. S. G.; ...
2016-07-25
Bionanoprobe (BNP), a hard x-ray fluorescence sample-scanning nanoprobe at the Advanced Photon Source of Argonne National Laboratory, has been used to quantitatively study elemental distributions in biological cells with sub-100 nm spatial resolution and high sensitivity. Cryogenic conditions enable biological samples to be studied in their frozen-hydrated state with both ultrastructure and elemental distributions more faithfully preserved compared to conventional chemical fixation or dehydration methods. Furthermore, radiation damage is reduced in two ways: the diffusion rate of free radicals is decreased at low temperatures; and the sample is embedded in vitrified ice, which reduces mass loss.
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A low temperature scanning force microscope for biological samples
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gustafsson, Mats Gustaf Lennart
1993-05-01
An SFM has been constructed capable of operating at 143 K. Two contributions to SFM technology are described: a new method of fabricating tips, and new designs of SFM springs that significantly lower the noise level. The SFM has been used to image several biological samples (including collagen, ferritin, RNA, purple membrane) at 143 K and room temperature. No improvement in resolution resulted from 143 K operation; several possible reasons for this are discussed. Possibly sharper tips may help. The 143 K SFM will allow the study of new categories of samples, such as those prepared by freeze-frame, single moleculesmore » (temperature dependence of mechanical properties), etc. The SFM was used to cut single collagen molecules into segments with a precision of {le} 10 nm.« less
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Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser.
Ekeberg, Tomas; Svenda, Martin; Seibert, M Marvin; Abergel, Chantal; Maia, Filipe R N C; Seltzer, Virginie; DePonte, Daniel P; Aquila, Andrew; Andreasson, Jakob; Iwan, Bianca; Jönsson, Olof; Westphal, Daniel; Odić, Duško; Andersson, Inger; Barty, Anton; Liang, Meng; Martin, Andrew V; Gumprecht, Lars; Fleckenstein, Holger; Bajt, Saša; Barthelmess, Miriam; Coppola, Nicola; Claverie, Jean-Michel; Loh, N Duane; Bostedt, Christoph; Bozek, John D; Krzywinski, Jacek; Messerschmidt, Marc; Bogan, Michael J; Hampton, Christina Y; Sierra, Raymond G; Frank, Matthias; Shoeman, Robert L; Lomb, Lukas; Foucar, Lutz; Epp, Sascha W; Rolles, Daniel; Rudenko, Artem; Hartmann, Robert; Hartmann, Andreas; Kimmel, Nils; Holl, Peter; Weidenspointner, Georg; Rudek, Benedikt; Erk, Benjamin; Kassemeyer, Stephan; Schlichting, Ilme; Strüder, Lothar; Ullrich, Joachim; Schmidt, Carlo; Krasniqi, Faton; Hauser, Günter; Reich, Christian; Soltau, Heike; Schorb, Sebastian; Hirsemann, Helmut; Wunderer, Cornelia; Graafsma, Heinz; Chapman, Henry; Hajdu, Janos
2016-08-01
Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.
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Frequency modulation atomic force microscopy: a dynamic measurement technique for biological systems
NASA Astrophysics Data System (ADS)
Higgins, Michael J.; Riener, Christian K.; Uchihashi, Takayuki; Sader, John E.; McKendry, Rachel; Jarvis, Suzanne P.
2005-03-01
Frequency modulation atomic force microscopy (FM-AFM) has been modified to operate in a liquid environment within an atomic force microscope specifically designed for investigating biological samples. We demonstrate the applicability of FM-AFM to biological samples using the spectroscopy mode to measure the unbinding forces of a single receptor-ligand (biotin-avidin) interaction. We show that quantitative adhesion force measurements can only be obtained provided certain modifications are made to the existing theory, which is used to convert the detected frequency shifts to an interaction force. Quantitative force measurements revealed that the unbinding forces for the biotin-avidin interaction were greater than those reported in previous studies. This finding was due to the use of high average tip velocities, which were calculated to be two orders of magnitude greater than those typically used in unbinding receptor-ligand experiments. This study therefore highlights the potential use of FM-AFM to study a range of biological systems, including living cells and/or single biomolecule interactions.
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Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser
NASA Astrophysics Data System (ADS)
Ekeberg, Tomas; Svenda, Martin; Seibert, M. Marvin; Abergel, Chantal; Maia, Filipe R. N. C.; Seltzer, Virginie; Deponte, Daniel P.; Aquila, Andrew; Andreasson, Jakob; Iwan, Bianca; Jönsson, Olof; Westphal, Daniel; Odić, Duško; Andersson, Inger; Barty, Anton; Liang, Meng; Martin, Andrew V.; Gumprecht, Lars; Fleckenstein, Holger; Bajt, Saša; Barthelmess, Miriam; Coppola, Nicola; Claverie, Jean-Michel; Loh, N. Duane; Bostedt, Christoph; Bozek, John D.; Krzywinski, Jacek; Messerschmidt, Marc; Bogan, Michael J.; Hampton, Christina Y.; Sierra, Raymond G.; Frank, Matthias; Shoeman, Robert L.; Lomb, Lukas; Foucar, Lutz; Epp, Sascha W.; Rolles, Daniel; Rudenko, Artem; Hartmann, Robert; Hartmann, Andreas; Kimmel, Nils; Holl, Peter; Weidenspointner, Georg; Rudek, Benedikt; Erk, Benjamin; Kassemeyer, Stephan; Schlichting, Ilme; Strüder, Lothar; Ullrich, Joachim; Schmidt, Carlo; Krasniqi, Faton; Hauser, Günter; Reich, Christian; Soltau, Heike; Schorb, Sebastian; Hirsemann, Helmut; Wunderer, Cornelia; Graafsma, Heinz; Chapman, Henry; Hajdu, Janos
2016-08-01
Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.
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Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.
Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun
2018-04-17
Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.
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Single mimivirus particles intercepted and imaged with an X-ray laser
Seibert, M. Marvin; Ekeberg, Tomas; Maia, Filipe R. N. C.; Svenda, Martin; Andreasson, Jakob; Jönsson, Olof; Odić, Duško; Iwan, Bianca; Rocker, Andrea; Westphal, Daniel; Hantke, Max; DePonte, Daniel P.; Barty, Anton; Schulz, Joachim; Gumprecht, Lars; Coppola, Nicola; Aquila, Andrew; Liang, Mengning; White, Thomas A.; Martin, Andrew; Caleman, Carl; Stern, Stephan; Abergel, Chantal; Seltzer, Virginie; Claverie, Jean-Michel; Bostedt, Christoph; Bozek, John D.; Boutet, Sébastien; Miahnahri, A. Alan; Messerschmidt, Marc; Krzywinski, Jacek; Williams, Garth; Hodgson, Keith O.; Bogan, Michael J.; Hampton, Christina Y.; Sierra, Raymond G.; Starodub, Dmitri; Andersson, Inger; Bajt, Saša; Barthelmess, Miriam; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Kirian, Richard; Hunter, Mark; Doak, R. Bruce; Marchesini, Stefano; Hau-Riege, Stefan P.; Frank, Matthias; Shoeman, Robert L.; Lomb, Lukas; Epp, Sascha W.; Hartmann, Robert; Rolles, Daniel; Rudenko, Artem; Schmidt, Carlo; Foucar, Lutz; Kimmel, Nils; Holl, Peter; Rudek, Benedikt; Erk, Benjamin; Hömke, André; Reich, Christian; Pietschner, Daniel; Weidenspointner, Georg; Strüder, Lothar; Hauser, Günter; Gorke, Hubert; Ullrich, Joachim; Schlichting, Ilme; Herrmann, Sven; Schaller, Gerhard; Schopper, Florian; Soltau, Heike; Kühnel, Kai-Uwe; Andritschke, Robert; Schröter, Claus-Dieter; Krasniqi, Faton; Bott, Mario; Schorb, Sebastian; Rupp, Daniela; Adolph, Marcus; Gorkhover, Tais; Hirsemann, Helmut; Potdevin, Guillaume; Graafsma, Heinz; Nilsson, Björn; Chapman, Henry N.; Hajdu, Janos
2014-01-01
X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions1–4. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma1. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval2. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source5. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies. PMID:21293374
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A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.
Haque, Ashraful; Engel, Jessica; Teichmann, Sarah A; Lönnberg, Tapio
2017-08-18
RNA sequencing (RNA-seq) is a genomic approach for the detection and quantitative analysis of messenger RNA molecules in a biological sample and is useful for studying cellular responses. RNA-seq has fueled much discovery and innovation in medicine over recent years. For practical reasons, the technique is usually conducted on samples comprising thousands to millions of cells. However, this has hindered direct assessment of the fundamental unit of biology-the cell. Since the first single-cell RNA-sequencing (scRNA-seq) study was published in 2009, many more have been conducted, mostly by specialist laboratories with unique skills in wet-lab single-cell genomics, bioinformatics, and computation. However, with the increasing commercial availability of scRNA-seq platforms, and the rapid ongoing maturation of bioinformatics approaches, a point has been reached where any biomedical researcher or clinician can use scRNA-seq to make exciting discoveries. In this review, we present a practical guide to help researchers design their first scRNA-seq studies, including introductory information on experimental hardware, protocol choice, quality control, data analysis and biological interpretation.
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Single-pulse enhanced coherent diffraction imaging of bacteria with an X-ray free-electron laser
NASA Astrophysics Data System (ADS)
Fan, Jiadong; Sun, Zhibin; Wang, Yaling; Park, Jaehyun; Kim, Sunam; Gallagher-Jones, Marcus; Kim, Yoonhee; Song, Changyong; Yao, Shengkun; Zhang, Jian; Zhang, Jianhua; Duan, Xiulan; Tono, Kensuke; Yabashi, Makina; Ishikawa, Tetsuya; Fan, Chunhai; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun; Earnest, Thomas; Jiang, Huaidong
2016-09-01
High-resolution imaging offers one of the most promising approaches for exploring and understanding the structure and function of biomaterials and biological systems. X-ray free-electron lasers (XFELs) combined with coherent diffraction imaging can theoretically provide high-resolution spatial information regarding biological materials using a single XFEL pulse. Currently, the application of this method suffers from the low scattering cross-section of biomaterials and X-ray damage to the sample. However, XFELs can provide pulses of such short duration that the data can be collected using the “diffract and destroy” approach before the effects of radiation damage on the data become significant. These experiments combine the use of enhanced coherent diffraction imaging with single-shot XFEL radiation to investigate the cellular architecture of Staphylococcus aureus with and without labeling by gold (Au) nanoclusters. The resolution of the images reconstructed from these diffraction patterns were twice as high or more for gold-labeled samples, demonstrating that this enhancement method provides a promising approach for the high-resolution imaging of biomaterials and biological systems.
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Single-pulse enhanced coherent diffraction imaging of bacteria with an X-ray free-electron laser
Fan, Jiadong; Sun, Zhibin; Wang, Yaling; Park, Jaehyun; Kim, Sunam; Gallagher-Jones, Marcus; Kim, Yoonhee; Song, Changyong; Yao, Shengkun; Zhang, Jian; Zhang, Jianhua; Duan, Xiulan; Tono, Kensuke; Yabashi, Makina; Ishikawa, Tetsuya; Fan, Chunhai; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun; Earnest, Thomas; Jiang, Huaidong
2016-01-01
High-resolution imaging offers one of the most promising approaches for exploring and understanding the structure and function of biomaterials and biological systems. X-ray free-electron lasers (XFELs) combined with coherent diffraction imaging can theoretically provide high-resolution spatial information regarding biological materials using a single XFEL pulse. Currently, the application of this method suffers from the low scattering cross-section of biomaterials and X-ray damage to the sample. However, XFELs can provide pulses of such short duration that the data can be collected using the “diffract and destroy” approach before the effects of radiation damage on the data become significant. These experiments combine the use of enhanced coherent diffraction imaging with single-shot XFEL radiation to investigate the cellular architecture of Staphylococcus aureus with and without labeling by gold (Au) nanoclusters. The resolution of the images reconstructed from these diffraction patterns were twice as high or more for gold-labeled samples, demonstrating that this enhancement method provides a promising approach for the high-resolution imaging of biomaterials and biological systems. PMID:27659203
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Single-pulse enhanced coherent diffraction imaging of bacteria with an X-ray free-electron laser.
Fan, Jiadong; Sun, Zhibin; Wang, Yaling; Park, Jaehyun; Kim, Sunam; Gallagher-Jones, Marcus; Kim, Yoonhee; Song, Changyong; Yao, Shengkun; Zhang, Jian; Zhang, Jianhua; Duan, Xiulan; Tono, Kensuke; Yabashi, Makina; Ishikawa, Tetsuya; Fan, Chunhai; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun; Earnest, Thomas; Jiang, Huaidong
2016-09-23
High-resolution imaging offers one of the most promising approaches for exploring and understanding the structure and function of biomaterials and biological systems. X-ray free-electron lasers (XFELs) combined with coherent diffraction imaging can theoretically provide high-resolution spatial information regarding biological materials using a single XFEL pulse. Currently, the application of this method suffers from the low scattering cross-section of biomaterials and X-ray damage to the sample. However, XFELs can provide pulses of such short duration that the data can be collected using the "diffract and destroy" approach before the effects of radiation damage on the data become significant. These experiments combine the use of enhanced coherent diffraction imaging with single-shot XFEL radiation to investigate the cellular architecture of Staphylococcus aureus with and without labeling by gold (Au) nanoclusters. The resolution of the images reconstructed from these diffraction patterns were twice as high or more for gold-labeled samples, demonstrating that this enhancement method provides a promising approach for the high-resolution imaging of biomaterials and biological systems.
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Optics-Integrated Microfluidic Platforms for Biomolecular Analyses
Bates, Kathleen E.; Lu, Hang
2016-01-01
Compared with conventional optical methods, optics implemented on microfluidic chips provide small, and often much cheaper ways to interrogate biological systems from the level of single molecules up to small model organisms. The optical probing of single molecules has been used to investigate the mechanical properties of individual biological molecules; however, multiplexing of these measurements through microfluidics and nanofluidics confers many analytical advantages. Optics-integrated microfluidic systems can significantly simplify sample processing and allow a more user-friendly experience; alignments of on-chip optical components are predetermined during fabrication and many purely optical techniques are passively controlled. Furthermore, sample loss from complicated preparation and fluid transfer steps can be virtually eliminated, a particularly important attribute for biological molecules at very low concentrations. Excellent fluid handling and high surface area/volume ratios also contribute to faster detection times for low abundance molecules in small sample volumes. Although integration of optical systems with classical microfluidic analysis techniques has been limited, microfluidics offers a ready platform for interrogation of biophysical properties. By exploiting the ease with which fluids and particles can be precisely and dynamically controlled in microfluidic devices, optical sensors capable of unique imaging modes, single molecule manipulation, and detection of minute changes in concentration of an analyte are possible. PMID:27119629
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Bioinformatics approaches to single-cell analysis in developmental biology.
Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H
2016-03-01
Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Big Results from Small Samples: Evaluation of Amplification Protocols for Gene Expression Profiling
Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed...
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duVerle, David A; Yotsukura, Sohiya; Nomura, Seitaro; Aburatani, Hiroyuki; Tsuda, Koji
2016-09-13
Single-cell RNA sequencing is fast becoming one the standard method for gene expression measurement, providing unique insights into cellular processes. A number of methods, based on general dimensionality reduction techniques, have been suggested to help infer and visualise the underlying structure of cell populations from single-cell expression levels, yet their models generally lack proper biological grounding and struggle at identifying complex differentiation paths. Here we introduce cellTree: an R/Bioconductor package that uses a novel statistical approach, based on document analysis techniques, to produce tree structures outlining the hierarchical relationship between single-cell samples, while identifying latent groups of genes that can provide biological insights. With cellTree, we provide experimentalists with an easy-to-use tool, based on statistically and biologically-sound algorithms, to efficiently explore and visualise single-cell RNA data. The cellTree package is publicly available in the online Bionconductor repository at: http://bioconductor.org/packages/cellTree/ .
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Wavefront correction using machine learning methods for single molecule localization microscopy
NASA Astrophysics Data System (ADS)
Tehrani, Kayvan F.; Xu, Jianquan; Kner, Peter
2015-03-01
Optical Aberrations are a major challenge in imaging biological samples. In particular, in single molecule localization (SML) microscopy techniques (STORM, PALM, etc.) a high Strehl ratio point spread function (PSF) is necessary to achieve sub-diffraction resolution. Distortions in the PSF shape directly reduce the resolution of SML microscopy. The system aberrations caused by the imperfections in the optics and instruments can be compensated using Adaptive Optics (AO) techniques prior to imaging. However, aberrations caused by the biological sample, both static and dynamic, have to be dealt with in real time. A challenge for wavefront correction in SML microscopy is a robust optimization approach in the presence of noise because of the naturally high fluctuations in photon emission from single molecules. Here we demonstrate particle swarm optimization for real time correction of the wavefront using an intensity independent metric. We show that the particle swarm algorithm converges faster than the genetic algorithm for bright fluorophores.
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Inferring Biological Structures from Super-Resolution Single Molecule Images Using Generative Models
Maji, Suvrajit; Bruchez, Marcel P.
2012-01-01
Localization-based super resolution imaging is presently limited by sampling requirements for dynamic measurements of biological structures. Generating an image requires serial acquisition of individual molecular positions at sufficient density to define a biological structure, increasing the acquisition time. Efficient analysis of biological structures from sparse localization data could substantially improve the dynamic imaging capabilities of these methods. Using a feature extraction technique called the Hough Transform simple biological structures are identified from both simulated and real localization data. We demonstrate that these generative models can efficiently infer biological structures in the data from far fewer localizations than are required for complete spatial sampling. Analysis at partial data densities revealed efficient recovery of clathrin vesicle size distributions and microtubule orientation angles with as little as 10% of the localization data. This approach significantly increases the temporal resolution for dynamic imaging and provides quantitatively useful biological information. PMID:22629348
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Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M T; Gradinaru, Viviana; Pierce, Niles A; Cai, Long
2016-08-01
Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. © 2016. Published by The Company of Biologists Ltd.
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Near-Infrared Fluorescent Materials for Sensing of Biological Targets
Amiot, Carrie L.; Xu, Shuping; Liang, Song; Pan, Lingyun; Zhao, Julia Xiaojun
2008-01-01
Near-infrared fluorescent (NIRF) materials are promising labeling reagents for sensitive determination and imaging of biological targets. In the near-infrared region biological samples have low background fluorescence signals, providing high signal to noise ratio. Meanwhile, near-infrared radiation can penetrate into sample matrices deeply due to low light scattering. Thus, in vivo and in vitro imaging of biological samples can be achieved by employing the NIRF probes. To take full advantage of NIRF materials in the biological and biomedical field, one of the key issues is to develop intense and biocompatible NIRF probes. In this review, a number of NIRF materials are discussed including traditional NIRF dye molecules, newly developed NIRF quantum dots and single-walled carbon nanotubes, as well as rare earth metal compounds. The use of some NIRF materials in various nanostructures is illustrated. The enhancement of NIRF using metal nanostructures is covered as well. The fluorescence mechanism and bioapplications of each type of the NIRF materials are discussed in details. PMID:27879867
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Ultrasonic characterization of single drops of liquids
Sinha, Dipen N.
1998-01-01
Ultrasonic characterization of single drops of liquids. The present invention includes the use of two closely spaced transducers, or one transducer and a closely spaced reflector plate, to form an interferometer suitable for ultrasonic characterization of droplet-size and smaller samples without the need for a container. The droplet is held between the interferometer elements, whose distance apart may be adjusted, by surface tension. The surfaces of the interferometer elements may be readily cleansed by a stream of solvent followed by purified air when it is desired to change samples. A single drop of liquid is sufficient for high-quality measurement. Examples of samples which may be investigated using the apparatus and method of the present invention include biological specimens (tear drops; blood and other body fluid samples; samples from tumors, tissues, and organs; secretions from tissues and organs; snake and bee venom, etc.) for diagnostic evaluation, samples in forensic investigations, and detection of drugs in small quantities.
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Yokota, Miyo
2005-05-01
In the United States, the biologically admixed population is increasing. Such demographic changes may affect the distribution of anthropometric characteristics, which are incorporated into the design of equipment and clothing for the US Army and other large organizations. The purpose of this study was to examine multivariate craniofacial anthropometric distributions between biologically admixed male populations and single racial groups of Black and White males. Multivariate statistical results suggested that nose breadth and lip length were different between Blacks and Whites. Such differences may be considered for adjustments to respirators and chemical-biological protective masks. However, based on this pilot study, multivariate anthropometric distributions of admixed individuals were within the distributions of single racial groups. Based on the sample reported, sizing and designing for the admixed groups are not necessary if anthropometric distributions of single racial groups comprising admixed groups are known.
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Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes.
Troell, Karin; Hallström, Björn; Divne, Anna-Maria; Alsmark, Cecilia; Arrighi, Romanico; Huss, Mikael; Beser, Jessica; Bertilsson, Stefan
2016-06-23
Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be easily maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. Cells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification. As a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units.
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NASA Technical Reports Server (NTRS)
Mitz, M. A.
1972-01-01
Some promising newer approaches for detecting microorganisms are discussed, giving particular attention to the integration of different methods into a single instrument. Life detection methods may be divided into biological, chemical, and cytological methods. Biological methods are based on the biological properties of assimilation, metabolism, and growth. Devices for the detection of organic materials are considered, taking into account an instrument which volatilizes, separates, and analyzes a sample sequentially. Other instrumental systems described make use of a microscope and the cytochemical staining principle.
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Luka, George; Ahmadi, Ali; Najjaran, Homayoun; Alocilja, Evangelyn; DeRosa, Maria; Wolthers, Kirsten; Malki, Ahmed; Aziz, Hassan; Althani, Asmaa; Hoorfar, Mina
2015-01-01
A biosensor can be defined as a compact analytical device or unit incorporating a biological or biologically derived sensitive recognition element immobilized on a physicochemical transducer to measure one or more analytes. Microfluidic systems, on the other hand, provide throughput processing, enhance transport for controlling the flow conditions, increase the mixing rate of different reagents, reduce sample and reagents volume (down to nanoliter), increase sensitivity of detection, and utilize the same platform for both sample preparation and detection. In view of these advantages, the integration of microfluidic and biosensor technologies provides the ability to merge chemical and biological components into a single platform and offers new opportunities for future biosensing applications including portability, disposability, real-time detection, unprecedented accuracies, and simultaneous analysis of different analytes in a single device. This review aims at representing advances and achievements in the field of microfluidic-based biosensing. The review also presents examples extracted from the literature to demonstrate the advantages of merging microfluidic and biosensing technologies and illustrate the versatility that such integration promises in the future biosensing for emerging areas of biological engineering, biomedical studies, point-of-care diagnostics, environmental monitoring, and precision agriculture. PMID:26633409
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Splitting a droplet for femtoliter liquid patterns and single cell isolation.
Li, Huizeng; Yang, Qiang; Li, Guannan; Li, Mingzhu; Wang, Shutao; Song, Yanlin
2015-05-06
Well-defined microdroplet generation has attracted great interest, which is important for the high-resolution patterning and matrix distribution for chemical reactions and biological assays. By sliding a droplet on a patterned superhydrophilic/superhydrophobic substrate, tiny microdroplet arrays low to femtoliter were achieved with uniform volume and composition. Using this method, cells were successfully isolated, resulting in a single cell array. The droplet-splitting method is facile, sample-effective, and low-cost, which will be of great potential for the development of microdroplet arrays for biological analysis as well as patterning system and devices.
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Universal nucleic acids sample preparation method for cells, spores and their mixture
Bavykin, Sergei [Darien, IL
2011-01-18
The present invention relates to a method for extracting nucleic acids from biological samples. More specifically the invention relates to a universal method for extracting nucleic acids from unidentified biological samples. An advantage of the presently invented method is its ability to effectively and efficiently extract nucleic acids from a variety of different cell types including but not limited to prokaryotic or eukaryotic cells and/or recalcitrant organisms (i.e. spores). Unlike prior art methods which are focused on extracting nucleic acids from vegetative cell or spores, the present invention effectively extracts nucleic acids from spores, multiple cell types or mixtures thereof using a single method. Important that the invented method has demonstrated an ability to extract nucleic acids from spores and vegetative bacterial cells with similar levels effectiveness. The invented method employs a multi-step protocol which erodes the cell structure of the biological sample, isolates, labels, fragments nucleic acids and purifies labeled samples from the excess of dye.
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Bayesian approach to MSD-based analysis of particle motion in live cells.
Monnier, Nilah; Guo, Syuan-Ming; Mori, Masashi; He, Jun; Lénárt, Péter; Bathe, Mark
2012-08-08
Quantitative tracking of particle motion using live-cell imaging is a powerful approach to understanding the mechanism of transport of biological molecules, organelles, and cells. However, inferring complex stochastic motion models from single-particle trajectories in an objective manner is nontrivial due to noise from sampling limitations and biological heterogeneity. Here, we present a systematic Bayesian approach to multiple-hypothesis testing of a general set of competing motion models based on particle mean-square displacements that automatically classifies particle motion, properly accounting for sampling limitations and correlated noise while appropriately penalizing model complexity according to Occam's Razor to avoid over-fitting. We test the procedure rigorously using simulated trajectories for which the underlying physical process is known, demonstrating that it chooses the simplest physical model that explains the observed data. Further, we show that computed model probabilities provide a reliability test for the downstream biological interpretation of associated parameter values. We subsequently illustrate the broad utility of the approach by applying it to disparate biological systems including experimental particle trajectories from chromosomes, kinetochores, and membrane receptors undergoing a variety of complex motions. This automated and objective Bayesian framework easily scales to large numbers of particle trajectories, making it ideal for classifying the complex motion of large numbers of single molecules and cells from high-throughput screens, as well as single-cell-, tissue-, and organism-level studies. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Martinez, Jennifer S [Santa Fe, NM; Swanson, Basil I [Los Alamos, NM; Shively, John E [Arcadia, CA; Li, Lin [Monrovia, CA
2009-06-02
An assay element is described including recognition ligands adapted for binding to carcinoembryonic antigen (CEA) bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of CEA is described including injecting a possible CEA-containing sample into a sensor cell including the assay element, maintaining the sample within the sensor cell for time sufficient for binding to occur between CEA present within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.
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Massively parallel digital transcriptional profiling of single cells
Zheng, Grace X. Y.; Terry, Jessica M.; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W.; Wilson, Ryan; Ziraldo, Solongo B.; Wheeler, Tobias D.; McDermott, Geoff P.; Zhu, Junjie; Gregory, Mark T.; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G.; Masquelier, Donald A.; Nishimura, Stefanie Y.; Schnall-Levin, Michael; Wyatt, Paul W.; Hindson, Christopher M.; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D.; Beppu, Lan W.; Deeg, H. Joachim; McFarland, Christopher; Loeb, Keith R.; Valente, William J.; Ericson, Nolan G.; Stevens, Emily A.; Radich, Jerald P.; Mikkelsen, Tarjei S.; Hindson, Benjamin J.; Bielas, Jason H.
2017-01-01
Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients. PMID:28091601
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NASA Astrophysics Data System (ADS)
Granero, Luis; Ferreira, Carlos; Zalevsky, Zeev; García, Javier; Micó, Vicente
2016-07-01
Single-Exposure Super-Resolved Interferometric Microscopy (SESRIM) reports on a way to achieve one-dimensional (1-D) superresolved imaging in digital holographic microscopy (DHM) by a single illumination shot and digital recording. SESRIM provides color-coded angular multiplexing of the accessible sample's range of spatial frequencies and it allows their recording in a single CCD (color or monochrome) snapshot by adding 3 RGB coherent reference beams at the output plane. In this manuscript, we extend the applicability of SESRIM to the field of digital in-line holographic microscopy (DIHM), that is, working without lenses. As consequence of the in-line configuration, an additional restriction concerning the object field of view (FOV) must be imposed to the technique. Experimental results are reported for both a synthetic object (USAF resolution test target) and a biological sample (swine sperm sample) validating this new kind of superresolution imaging method named as lensless SESRIM (L-SESRIM).
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High Resolution Tissue Imaging Using the Single-probe Mass Spectrometry under Ambient Conditions
NASA Astrophysics Data System (ADS)
Rao, Wei; Pan, Ning; Yang, Zhibo
2015-06-01
Ambient mass spectrometry imaging (MSI) is an emerging field with great potential for the detailed spatial analysis of biological samples with minimal pretreatment. We have developed a miniaturized sampling and ionization device, the Single-probe, which uses in-situ surface micro-extraction to achieve high detection sensitivity and spatial resolution during MSI experiments. The Single-probe was coupled to a Thermo LTQ Orbitrap XL mass spectrometer and was able to create high spatial and high mass resolution MS images at 8 ± 2 and 8.5 μm on flat polycarbonate microscope slides and mouse kidney sections, respectively, which are among the highest resolutions available for ambient MSI techniques. Our proof-of-principle experiments indicate that the Single-probe MSI technique has the potential to obtain ambient MS images with very high spatial resolutions with minimal sample preparation, which opens the possibility for subcellular ambient tissue MSI to be performed in the future.
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Fast Raman single bacteria identification: toward a routine in-vitro diagnostic
NASA Astrophysics Data System (ADS)
Douet, Alice; Josso, Quentin; Marchant, Adrien; Dutertre, Bertrand; Filiputti, Delphine; Novelli-Rousseau, Armelle; Espagnon, Isabelle; Kloster-Landsberg, Meike; Mallard, Frédéric; Perraut, Francois
2016-04-01
Timely microbiological results are essential to allow clinicians to optimize the prescribed treatment, ideally at the initial stage of the therapeutic process. Several approaches have been proposed to solve this issue and to provide the microbiological result in a few hours directly from the sample such as molecular biology. However fast and sensitive those methods are not based on single phenotypic information which presents several drawbacks and limitations. Optical methods have the advantage to allow single-cell sensitivity and to probe the phenotype of measured cells. Here we present a process and a prototype that allow automated single-bacteria phenotypic analysis. This prototype is based on the use of Digital In-line Holography techniques combined with a specially designed Raman spectrometer using a dedicated device to capture bacteria. The localization of single-cell is finely determined by using holograms and a proper propagation kernel. Holographic images are also used to analyze bacteria in the sample to sort potential pathogens from flora dwelling species or other biological particles. This accurate localization enables the use of a small confocal volume adapted to the measurement of single-cell. Along with the confocal volume adaptation, we also have modified every components of the spectrometer to optimize single-bacteria Raman measurements. This optimization allowed us to acquire informative single-cell spectra using an integration time of 0.5s only. Identification results obtained with this prototype are presented based on a 65144 Raman spectra database acquired automatically on 48 bacteria strains belonging to 8 species.
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Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin
2017-03-01
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
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Ultrasonic characterization of single drops of liquids
Sinha, D.N.
1998-04-14
Ultrasonic characterization of single drops of liquids is disclosed. The present invention includes the use of two closely spaced transducers, or one transducer and a closely spaced reflector plate, to form an interferometer suitable for ultrasonic characterization of droplet-size and smaller samples without the need for a container. The droplet is held between the interferometer elements, whose distance apart may be adjusted, by surface tension. The surfaces of the interferometer elements may be readily cleansed by a stream of solvent followed by purified air when it is desired to change samples. A single drop of liquid is sufficient for high-quality measurement. Examples of samples which may be investigated using the apparatus and method of the present invention include biological specimens (tear drops; blood and other body fluid samples; samples from tumors, tissues, and organs; secretions from tissues and organs; snake and bee venom, etc.) for diagnostic evaluation, samples in forensic investigations, and detection of drugs in small quantities. 5 figs.
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Ultrasonic characterization of single drops of liquids
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sinha, D.N.
Ultrasonic characterization of single drops of liquids is disclosed. The present invention includes the use of two closely spaced transducers, or one transducer and a closely spaced reflector plate, to form an interferometer suitable for ultrasonic characterization of droplet-size and smaller samples without the need for a container. The droplet is held between the interferometer elements, whose distance apart may be adjusted, by surface tension. The surfaces of the interferometer elements may be readily cleansed by a stream of solvent followed by purified air when it is desired to change samples. A single drop of liquid is sufficient for high-qualitymore » measurement. Examples of samples which may be investigated using the apparatus and method of the present invention include biological specimens (tear drops; blood and other body fluid samples; samples from tumors, tissues, and organs; secretions from tissues and organs; snake and bee venom, etc.) for diagnostic evaluation, samples in forensic investigations, and detection of drugs in small quantities. 5 figs.« less
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Use of single scatter electron monte carlo transport for medical radiation sciences
Svatos, Michelle M.
2001-01-01
The single scatter Monte Carlo code CREEP models precise microscopic interactions of electrons with matter to enhance physical understanding of radiation sciences. It is designed to simulate electrons in any medium, including materials important for biological studies. It simulates each interaction individually by sampling from a library which contains accurate information over a broad range of energies.
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Group refractive index reconstruction with broadband interferometric confocal microscopy
Marks, Daniel L.; Schlachter, Simon C.; Zysk, Adam M.; Boppart, Stephen A.
2010-01-01
We propose a novel method of measuring the group refractive index of biological tissues at the micrometer scale. The technique utilizes a broadband confocal microscope embedded into a Mach–Zehnder interferometer, with which spectral interferograms are measured as the sample is translated through the focus of the beam. The method does not require phase unwrapping and is insensitive to vibrations in the sample and reference arms. High measurement stability is achieved because a single spectral interferogram contains all the information necessary to compute the optical path delay of the beam transmitted through the sample. Included are a physical framework defining the forward problem, linear solutions to the inverse problem, and simulated images of biologically relevant phantoms. PMID:18451922
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Farash, Katherine; Hanson, Erin K; Ballantyne, Jack
2015-03-09
DNA profiles can be obtained from 'touch DNA' evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a 'blind-swabbing' approach will co-sample cellular material from the different individuals, even if the individuals' cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim's DNA may be found in significant excess thus masking any potential perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, 'smart analysis' method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., "clumps") bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.
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Petegrosso, Raphael; Tolar, Jakub
2018-01-01
Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC. PMID:29630593
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Chavez, Rosa A; Valdivia, Isabel M; Oliva, Marcelo E
2007-06-01
Parasites have been used successfully as biological tags in population studies, mainly in marine fishes, but also in marine mammals, crustaceans and molluscs. Almost all published information dealing with parasites as biological tags evaluates differences between localities. However, local variability in the component community has not been assessed. In this work, we examined whether local variation of the metazoan parasite fauna of Engraulis ringens, extracted from five independent samples from two nearby localities in northern Chile, can be a factor causing bias in stock identification. Our results show that local variability, as estimated by a single sample, may suffice to represent component community variability with no need for replicated data.
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Development of Magnetic Nanomaterials and Devices for Biological Applications
2007-10-30
analysis. Suitable crystals for the X-ray diffraction analysis were grown as dark red plates from a saturated hexane solution of [ Co3 (CO)9CCH3] at 4 ºC...Commercially available magnetic nanoparticles are suitable for cell separation where a large number of particles are used to separate a single cell...from a sample. The magnetic moment of these particles is not high enough to enable the separation of single antigen molecules using a single particle
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NASA Astrophysics Data System (ADS)
Zivkovic, Sanja; Momcilovic, Milos; Staicu, Angela; Mutic, Jelena; Trtica, Milan; Savovic, Jelena
2017-02-01
The aim of this study was to develop a simple laser induced breakdown spectroscopy (LIBS) method for quantitative elemental analysis of powdered biological materials based on laboratory prepared calibration samples. The analysis was done using ungated single pulse LIBS in ambient air at atmospheric pressure. Transversely-Excited Atmospheric pressure (TEA) CO2 laser was used as an energy source for plasma generation on samples. The material used for the analysis was a blue-green alga Spirulina, widely used in food and pharmaceutical industries and also in a few biotechnological applications. To demonstrate the analytical potential of this particular LIBS system the obtained spectra were compared to the spectra obtained using a commercial LIBS system based on pulsed Nd:YAG laser. A single sample of known concentration was used to estimate detection limits for Ba, Ca, Fe, Mg, Mn, Si and Sr and compare detection power of these two LIBS systems. TEA CO2 laser based LIBS was also applied for quantitative analysis of the elements in powder Spirulina samples. Analytical curves for Ba, Fe, Mg, Mn and Sr were constructed using laboratory produced matrix-matched calibration samples. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used as the reference technique for elemental quantification, and reasonably well agreement between ICP and LIBS data was obtained. Results confirm that, in respect to its sensitivity and precision, TEA CO2 laser based LIBS can be successfully applied for quantitative analysis of macro and micro-elements in algal samples. The fact that nearly all classes of materials can be prepared as powders implies that the proposed method could be easily extended to a quantitative analysis of different kinds of materials, organic, biological or inorganic.
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Analytical Chemistry in Microenvironments: Single Nerve Cells.
1992-03-16
length of the capillary (34). Electroosmotic flow offers three key advantages for separation of small biological samples. First, this flow, if not...from microenvironments (ie. single cells). Indeed, volumes as low as 270 femtoliters have been injected using electroosmotic flow (15). Finally... electroosmotic flow provides a flat flow profile, since there is no stationary support between the origin of flow (capillary wall) and the bulk of solution
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Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng
2017-01-01
The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.
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Statistical inference from multiple iTRAQ experiments without using common reference standards.
Herbrich, Shelley M; Cole, Robert N; West, Keith P; Schulze, Kerry; Yager, James D; Groopman, John D; Christian, Parul; Wu, Lee; O'Meally, Robert N; May, Damon H; McIntosh, Martin W; Ruczinski, Ingo
2013-02-01
Isobaric tags for relative and absolute quantitation (iTRAQ) is a prominent mass spectrometry technology for protein identification and quantification that is capable of analyzing multiple samples in a single experiment. Frequently, iTRAQ experiments are carried out using an aliquot from a pool of all samples, or "masterpool", in one of the channels as a reference sample standard to estimate protein relative abundances in the biological samples and to combine abundance estimates from multiple experiments. In this manuscript, we show that using a masterpool is counterproductive. We obtain more precise estimates of protein relative abundance by using the available biological data instead of the masterpool and do not need to occupy a channel that could otherwise be used for another biological sample. In addition, we introduce a simple statistical method to associate proteomic data from multiple iTRAQ experiments with a numeric response and show that this approach is more powerful than the conventionally employed masterpool-based approach. We illustrate our methods using data from four replicate iTRAQ experiments on aliquots of the same pool of plasma samples and from a 406-sample project designed to identify plasma proteins that covary with nutrient concentrations in chronically undernourished children from South Asia.
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Lo, Shih-Jie; Yao, Da-Jeng
2015-07-23
This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.
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Lo, Shih-Jie; Yao, Da-Jeng
2015-01-01
This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918
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Design and Analysis of Single-Cell Sequencing Experiments.
Grün, Dominic; van Oudenaarden, Alexander
2015-11-05
Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.
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Near-infrared Raman spectroscopy of single optically trapped biological cells
NASA Astrophysics Data System (ADS)
Xie, Changan; Dinno, Mumtaz A.; Li, Yong-Qing
2002-02-01
We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.
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Multichannel waveguides for the simultaneous detection of disease biomarkers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mukundan, Harshini; Price, Dominique Z; Grace, Wynne K
2009-01-01
The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor that has previously been used for the detection of biomarkers associated with diseases such as tuberculosis, breast cancer, anthrax and influenza in complex biological samples (e.g., serum and urine). However, no single biomarker can accurately predict disease. To address this issue, we developed a multiplex assay for the detection of components of the Bacillus anthracis lethal toxin on single mode planar optical waveguides with tunable quantum dots as the fluorescence reporter. This limited ability to multiplex is still insufficient for accurate detection of disease ormore » for monitoring prognosis. In this manuscript, we demonstrate for the first time, the design, fabrication and successful evaluation of a multichannel planar optical waveguide for the simultaneous detection of at least three unknown samples in quadruplicate. We demonstrate the simultaneous, rapid (30 min), quantitative (with internal standard) and sensitive (limit of detection of 1 pM) detection of protective antigen and lethal factor of Bacillus anthracis in complex biological samples (serum) using specific monoclonal antibodies labeled with quantum dots as the fluorescence reporter.« less
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Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations
Mlodzianoski, Michael J.; Curthoys, Nikki M.; Gunewardene, Mudalige S.; Carter, Sean; Hess, Samuel T.
2016-01-01
Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample. PMID:27002724
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C.
2016-05-03
ABSTRACT Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. Themetabolite,protein, andlipidextraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of thismore » protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental,in vitro, and clinical). IMPORTANCEIn systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample.« less
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Estimating lifetime risk from spot biomarker data and intra‐class correlation coefficients (ICC)
Human biomarker measurements in tissues including blood, breath, and urine can serve as efficient surrogates for environmental monitoring because a single biological sample integrates personal exposure across all environmental media and uptake pathways. However, biomarkers repres...
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Assessing the bioavailability and risk from metal-contaminated soils and dusts
Exposure to contaminated soil and dust is an important pathway in human health risk assessment. Physical and chemical characteristics, as well as biological factors, determine the bioaccessibility/bioavailability of soil and dust contaminants. Within a single sample, contaminat...
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Integrated experimental platforms to study blast injuries: a bottom-up approach
NASA Astrophysics Data System (ADS)
Bo, C.; Williams, A.; Rankin, S.; Proud, W. G.; Brown, K. A.
2014-05-01
We are developing experimental models of blast injury using data from live biological samples. An integrated research strategy is followed to study material and biological properties of cells, tissues and organs, that are subjected to dynamic and static pressures, relevant to those of battlefield blast. We have developed a confined Split Hopkinson Pressure Bar (SHPB) system, which allows cells, either in suspension or as a monolayer, to be subjected to compression waves with pressures on the order of a few MPa and durations of hundreds of microseconds. The chamber design enables recovery of biological samples for cellular and molecular analysis. The SHPB platform, coupled with Quasi-Static experiments, is used to determine stress-strain curves of soft biological tissues under compression at low, medium and high strain rates. Tissue samples are examined, using histological techniques, to study macro- and microscopic changes induced by compression waves. In addition, a shock tube enables application of single or multiple air blasts with pressures on the order of kPa and a few milliseconds duration; this platform was used for initial studies on mesenchymal stem cells responses to blast pressures.
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Thickness determination of biological samples with a zeta-calibrated scanning tunneling microscope.
Wang, Z H; Hartmann, T; Baumeister, W; Guckenberger, R
1990-01-01
A single-tube scanning tunneling microscope has been zeta-calibrated by using atomic steps of crystalline gold and was used for measuring the thickness of two biological samples, metal-coated as well as uncoated. The hexagonal surface layer of the bacterium Deinococcus radiodurans with an open network-type structure shows thickness values that are strongly influenced by the substrate and the preparation method. In contrast, the thickness of the purple membrane of Halobacterium halobium with its densely packed less-corrugated structure exhibits very little variation in thickness in coated preparations and the values obtained are in good agreement with x-ray data. Images PMID:2251276
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2014-09-30
Caught and Released vs. Single Stranded Marine Mammals Michael Moore Biology Department Woods Hole Oceanographic Institution Woods Hole, MA 02543...analyze blood samples from captive, wild-caught, and stranded marine mammals in order to compare concentrations of Microparticles (MPs). If confirmed...military sonar or during seismic exploration, may harm marine animals. It has been suggested that alteration in physiology or diving behavior may
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Superresolution Imaging using Single-Molecule Localization
Patterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, Jennifer
2013-01-01
Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (∼10–20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales. PMID:20055680
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Zhong, Bineng; Pan, Shengnan; Zhang, Hongbo; Wang, Tian; Du, Jixiang; Chen, Duansheng; Cao, Liujuan
2016-01-01
In this paper, we propose deep architecture to dynamically learn the most discriminative features from data for both single-cell and object tracking in computational biology and computer vision. Firstly, the discriminative features are automatically learned via a convolutional deep belief network (CDBN). Secondly, we design a simple yet effective method to transfer features learned from CDBNs on the source tasks for generic purpose to the object tracking tasks using only limited amount of training data. Finally, to alleviate the tracker drifting problem caused by model updating, we jointly consider three different types of positive samples. Extensive experiments validate the robustness and effectiveness of the proposed method.
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Pan, Shengnan; Zhang, Hongbo; Wang, Tian; Du, Jixiang; Chen, Duansheng; Cao, Liujuan
2016-01-01
In this paper, we propose deep architecture to dynamically learn the most discriminative features from data for both single-cell and object tracking in computational biology and computer vision. Firstly, the discriminative features are automatically learned via a convolutional deep belief network (CDBN). Secondly, we design a simple yet effective method to transfer features learned from CDBNs on the source tasks for generic purpose to the object tracking tasks using only limited amount of training data. Finally, to alleviate the tracker drifting problem caused by model updating, we jointly consider three different types of positive samples. Extensive experiments validate the robustness and effectiveness of the proposed method. PMID:27847827
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Differential Multiphoton Laser Scanning Microscopy
Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.
2016-01-01
Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot. PMID:27390511
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Lippok, Norman; Villiger, Martin; Jun, Chang–Su; Bouma, Brett E.
2015-01-01
Fiber–based polarization sensitive OFDI is more challenging than free–space implementations. Using multiple input states, fiber–based systems provide sample birefringence information with the benefit of a flexible sample arm but come at the cost of increased system and acquisition complexity, and either reduce acquisition speed or require increased acquisition bandwidth. Here we show that with the calibration of a single polarization state, fiber–based configurations can approach the conceptual simplicity of traditional free–space configurations. We remotely control the polarization state of the light incident at the sample using the eigenpolarization states of a wave plate as a reference, and determine the Jones matrix of the output fiber. We demonstrate this method for polarization sensitive imaging of biological samples. PMID:25927775
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Refractive index measurements of single, spherical cells using digital holographic microscopy.
Schürmann, Mirjam; Scholze, Jana; Müller, Paul; Chan, Chii J; Ekpenyong, Andrew E; Chalut, Kevin J; Guck, Jochen
2015-01-01
In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue or light-cell interactions. Hence, determining the refractive index of cells using DHM is valuable in many biological applications. This chapter covers the main topics that are important for the implementation of DHM: setup, sample preparation, and analysis. First, the optical setup is described in detail including notes and suggestions for the implementation. Following that, a protocol for the sample and measurement preparation is explained. In the analysis section, an algorithm for the determination of quantitative phase maps is described. Subsequently, all intermediate steps for the calculation of the refractive index of suspended cells are presented, exploiting their spherical shape. In the last section, a discussion of possible extensions to the setup, further measurement configurations, and additional analysis methods are given. Throughout this chapter, we describe a simple, robust, and thus easily reproducible implementation of DHM. The different possibilities for extensions show the diverse fields of application for this technique. Copyright © 2015 Elsevier Inc. All rights reserved.
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Drone Transport of Chemistry and Hematology Samples Over Long Distances.
Amukele, Timothy K; Hernandez, James; Snozek, Christine L H; Wyatt, Ryan G; Douglas, Matthew; Amini, Richard; Street, Jeff
2017-11-02
We addressed the stability of biological samples in prolonged drone flights by obtaining paired chemistry and hematology samples from 21 adult volunteers in a single phlebotomy event-84 samples total. Half of the samples were held stationary, while the other samples were flown for 3 hours (258 km) in a custom active cooling box mounted on the drone. After the flight, 19 chemistry and hematology tests were performed. Seventeen analytes had small or no bias, but glucose and potassium in flown samples showed an 8% and 6.2% bias, respectively. The flown samples (mean, 24.8°C) were a mean of 2.5°C cooler than the stationary samples (mean, 27.3°C) during transportation to the flight field as well as during the flight. The changes in glucose and potassium are consistent with the magnitude and duration of the temperature difference between the flown and stationary samples. Long drone flights of biological samples are feasible but require stringent environmental controls to ensure consistent results. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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Berglund, E. Carina; Kuklinski, Nicholas J.; Karagündüz, Ekin; Ucar, Kubra; Hanrieder, Jörg; Ewing, Andrew G.
2013-01-01
Micellar electrokinetic capillary chromatography with electrochemical detection has been used to quantify biogenic amines in freeze-dried Drosophila melanogaster brains. Freeze drying samples offers a way to preserve the biological sample while making dissection of these tiny samples easier and faster. Fly samples were extracted in cold acetone and dried in a rotary evaporator. Extraction and drying times were optimized in order to avoid contamination by red-pigment from the fly eyes and still have intact brain structures. Single freeze-dried fly-brain samples were found to produce representative electropherograms as a single hand-dissected brain sample. Utilizing the faster dissection time that freeze drying affords, the number of brains in a fixed homogenate volume can be increased to concentrate the sample. Thus, concentrated brain samples containing five or fifteen preserved brains were analyzed for their neurotransmitter content, and five analytes; dopamine N-acetyloctopamine, Nacetylserotonin, N-acetyltyramine, N-acetyldopamine were found to correspond well with previously reported values. PMID:23387977
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Programmable lab-on-a-chip system for single cell analysis
NASA Astrophysics Data System (ADS)
Thalhammer, S.
2009-05-01
The collection, selection, amplification and detection of minimum genetic samples became a part of everyday life in medical and biological laboratories, to analyze DNA-fragments of pathogens, patient samples and traces on crime scenes. About a decade ago, a handful of researchers began discussing an intriguing idea. Could the equipment needed for everyday chemistry and biology procedures be shrunk to fit on a chip in the size of a fingernail? Miniature devices for, say, analysing DNA and proteins should be faster and cheaper than conventional versions. Lab-on-a-chip is an advanced technology that integrates a microfluidic system on a microscale chip device. The "laboratory" is created by means of channels, mixers, reservoirs, diffusion chambers, integrated electrodes, pumps, valves and more. With lab-ona- chip technology, complete laboratories on a square centimetre can be created. Here, a multifunctional programmable Lab-on-a-Chip driven by nanofluidics and controlled by surface acoustic waves (SAW) is presented. This system combines serial DNA-isolation-, amplification- and array-detection-process on a modified glass-platform. The fluid actuation is controlled via SAW by interdigital transducers implemented in the chemical modified chip surface. The chemical surface modification allows fluid handling in the sub-microliter range. Minute amount of sample material is extracted by laser-based microdissection out of e.g. histological sections at the single cell level. A few picogram of genetic material are isolated and transferred via a low-pressure transfer system (SPATS) onto the chip. Subsequently the genetic material inside single droplets, which behave like "virtual" beaker, is transported to the reaction and analysis centers on the chip surface via surface acoustic waves, mainly known as noise dumping filters in mobile phones. At these "biological reactors" the genetic material is processed, e.g. amplified via polymerase chain reaction methods, and genetically characterized.
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Emerging opportunities in structural biology with X-ray free-electron lasers
Schlichting, Ilme; Miao, Jianwei
2012-01-01
X-ray free-electron lasers (X-FELs) produce X-ray pulses with extremely brilliant peak intensity and ultrashort pulse duration. It has been proposed that radiation damage can be “outrun” by using an ultra intense and short X-FEL pulse that passes a biological sample before the onset of significant radiation damage. The concept of “diffraction-before-destruction” has been demonstrated recently at the Linac Coherent Light Source, the first operational hard X-ray FEL, for protein nanocrystals and giant virus particles. The continuous diffraction patterns from single particles allow solving the classical “phase problem” by the oversampling method with iterative algorithms. If enough data are collected from many identical copies of a (biological) particle, its three-dimensional structure can be reconstructed. We review the current status and future prospects of serial femtosecond crystallography (SFX) and single-particle coherent diffraction imaging (CDI) with X-FELs. PMID:22922042
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NASA Astrophysics Data System (ADS)
Prukner, Vaclav; Dolezalova, Eva; Simek, Milan
2014-10-01
Highly reactive environment produced by atmospheric-pressure, non-equilibrium plasmas generated by surface dielectric barrier discharges (SDBDs) may be used for inactivation of biologically contaminated surfaces. We investigated decontamination efficiency of reactive environment produced by single/multiple surface streamer micro-discharge driven by amplitude-modulated AC power in coplanar electrode geometry on biologically contaminated surface by Escherichia coli. The discharges were fed by synthetic air with water vapor admixtures at atmospheric pressure, time of treatment was set from 10 second to 10 minutes, diameters of used SDBD electrodes (single and multiple streamer) and homogeneously contaminated disc samples were equal (25 mm), the distance between the electrode and contaminated surface was 2 mm. Both a conventional cultivation and fluorescent method LIVE/DEAD Bacterial Viability kit were applied to estimate counts of bacteria after the plasma treatment. Inactivation was effective and bacteria partly lost ability to grow and became injured and viable/active but non-cultivable (VBNC/ABNC). Work was supported by the MEYS under Project LD13010, VES13 COST CZ (COST Action MP 1101).
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Determination of optical coefficients of biological tissue from a single integrating-sphere
NASA Astrophysics Data System (ADS)
Zhang, Lianshun; Shi, Aijuan; Lu, Hongguang
2012-01-01
The detection of interactions between light and tissue can be used to characterize the optical properties of the tissue. The development is described of a method that determines optical coefficients of biological tissue from a single optical reflectance spectrum measured with an integrating-sphere. The experimental system incorporated a DH-2000 deuterium tungsten halogen light source, a USB4000-VIS-NIR miniature fiber optic spectrometer and an integrating-sphere. Fat emulsion and ink were used to mimic the scattering and absorbing properties of tissue in the tested sample. The measured optical reflectance spectrums with different scattering and absorbing properties were used to train a back-propagation neural network (BPNN). Then the neural network (BPNN) was used to determine the optical coefficients of biological tissue from a single optical reflectance spectrum measured with an integrating-sphere. Tests on tissue-simulation phantoms showed the relative errors of this technique to be 7% for the reduced scattering coefficient and 15% for the absorption coefficients. The optical properties of human skin were also measured in vivo.
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NASA Astrophysics Data System (ADS)
Mandula, Ondrej; Allier, Cédric; Hervé, Lionel; Denarier, Eric; Fourest-Lieuvin, Anne; Gory-Fauré, Sylvie; Vinit, Angélique; Morales, Sophie
2018-02-01
We present a simple and compact phase imaging microscope for long-term observation of non-absorbing biological samples such as unstained cells in nutritive media. The phase image is obtained from a single defocused image taken with a standard wide-field microscope. Using a semi-coherent light source allows us to computationally re-focus image post-acquisition and recover both phase and transmission of the complex specimen. The simplicity of the system reduces both the cost and its physical size and allows a long-term observation of samples directly in a standard biological incubator. The low cost of the system can contribute to the democratization of science by allowing to perform complex long-term biological experiments to the laboratories with constrained budget. In this proceeding we present several results taken with our prototype and discuss the possibilities and limitations of our system.
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Trépout, Sylvain; Bastin, Philippe; Marco, Sergio
2017-03-12
This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.
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Potentials of single-cell biology in identification and validation of disease biomarkers.
Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong
2016-09-01
Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
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High-resolution, high-throughput imaging with a multibeam scanning electron microscope.
Eberle, A L; Mikula, S; Schalek, R; Lichtman, J; Knothe Tate, M L; Zeidler, D
2015-08-01
Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.
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NASA Astrophysics Data System (ADS)
Lagus, Todd P.; Edd, Jon F.
2013-03-01
Most cell biology experiments are performed in bulk cell suspensions where cell secretions become diluted and mixed in a contiguous sample. Confinement of single cells to small, picoliter-sized droplets within a continuous phase of oil provides chemical isolation of each cell, creating individual microreactors where rare cell qualities are highlighted and otherwise undetectable signals can be concentrated to measurable levels. Recent work in microfluidics has yielded methods for the encapsulation of cells in aqueous droplets and hydrogels at kilohertz rates, creating the potential for millions of parallel single-cell experiments. However, commercial applications of high-throughput microdroplet generation and downstream sensing and actuation methods are still emerging for cells. Using fluorescence-activated cell sorting (FACS) as a benchmark for commercially available high-throughput screening, this focused review discusses the fluid physics of droplet formation, methods for cell encapsulation in liquids and hydrogels, sensors and actuators and notable biological applications of high-throughput single-cell droplet microfluidics.
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Atomic Force Microscopy of Biological Membranes
Frederix, Patrick L.T.M.; Bosshart, Patrick D.; Engel, Andreas
2009-01-01
Abstract Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes. PMID:19167286
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Farash, Katherine; Hanson, Erin K.; Ballantyne, Jack
2015-01-01
DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material. PMID:25867046
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Single cell digital polymerase chain reaction on self-priming compartmentalization chip
Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying
2017-01-01
Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%–4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease. PMID:28191267
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Single cell digital polymerase chain reaction on self-priming compartmentalization chip.
Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying
2017-01-01
Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%-4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease.
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Compressive Force Spectroscopy: From Living Cells to Single Proteins.
Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark
2018-03-23
One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.
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Chavshin, Ali Reza; Oshaghi, Mohammad Ali; Vatandoost, Hasan; Hanafi-Bojd, Ahmad Ali; Raeisi, Ahmad; Nikpoor, Fatemeh
2014-01-01
Objective To identify the biological forms, sporozoite rate and molecular characterization of the Anopheles stephensi (An. stephensi) in Hormozgan and Sistan-Baluchistan provinces, the most important malarious areas in Iran. Methods Wild live An. stephensi samples were collected from different malarious areas in southern Iran. The biological forms were identified based on number of egg-ridges. Molecular characterization of biological forms was verified by analysis of the mitochondrial cytochrome oxidase subunit I and II (mtDNA-COI/COII). The Plasmodium infection was examined in the wild female specimens by species-specific nested–PCR method. Results Results showed that all three biological forms including mysorensis, intermediate and type are present in the study areas. Molecular investigations revealed no genetic variation between mtDNA COI/COII sequences of the biological forms and no Plasmodium parasites was detected in the collected mosquito samples. Conclusions Presence of three biological forms with identical sequences showed that the known biological forms belong to a single taxon and the various vectorial capacities reported for these forms are more likely corresponded to other epidemiological factors than to the morphotype of the populations. Lack of malaria parasite infection in An. stephensi, the most important vector of malaria, may be partly due to the success and achievement of ongoing active malaria control program in the region. PMID:24144130
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Towards single particle imaging of human chromosomes at SACLA
NASA Astrophysics Data System (ADS)
Robinson, Ian; Schwenke, Joerg; Yusuf, Mohammed; Estandarte, Ana; Zhang, Fucai; Chen, Bo; Clark, Jesse; Song, Changyong; Nam, Daewoong; Joti, Yasumasa; Tono, Kensuke; Yabashi, Makina; Ratnasari, Gina; Kaneyoshi, Kohei; Takata, Hideaki; Fukui, Kiichi
2015-12-01
Single particle imaging (SPI) is one of the front-page opportunities which were used to motivate the construction of the first x-ray free electron lasers (XFELs). SPI’s big advantage is that it avoids radiation damage to biological samples because the diffraction takes place in femtosecond single shots before any atomic motion can take place in the sample, hence before the onset of radiation damage. This is the ‘diffract before destruction’ theme, destruction being assured from the high x-ray doses used. This article reports our collaboration’s first attempt at SPI using the SACLA XFEL facility in June 2015. The report is limited to experience with the instrumentation and examples of data because we have not yet had time to invert them to images.
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Matsunaga, Hiroko; Goto, Mari; Arikawa, Koji; Shirai, Masataka; Tsunoda, Hiroyuki; Huang, Huan; Kambara, Hideki
2015-02-15
Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes. Copyright © 2014 Elsevier Inc. All rights reserved.
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Single-image diffusion coefficient measurements of proteins in free solution.
Zareh, Shannon Kian; DeSantis, Michael C; Kessler, Jonathan M; Li, Je-Luen; Wang, Y M
2012-04-04
Diffusion coefficient measurements are important for many biological and material investigations, such as studies of particle dynamics and kinetics, and size determinations. Among current measurement methods, single particle tracking (SPT) offers the unique ability to simultaneously obtain location and diffusion information about a molecule while using only femtomoles of sample. However, the temporal resolution of SPT is limited to seconds for single-color-labeled samples. By directly imaging three-dimensional diffusing fluorescent proteins and studying the widths of their intensity profiles, we were able to determine the proteins' diffusion coefficients using single protein images of submillisecond exposure times. This simple method improves the temporal resolution of diffusion coefficient measurements to submilliseconds, and can be readily applied to a range of particle sizes in SPT investigations and applications in which diffusion coefficient measurements are needed, such as reaction kinetics and particle size determinations. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Lee, Seunghyun; Kim, Hyemin; Shin, Seungjun; Doh, Junsang; Kim, Chulhong
2017-03-01
Optical microscopy (OM) and photoacoustic microscopy (PAM) have previously been used to image the optical absorption of intercellular features of biological cells. However, the optical diffraction limit ( 200 nm) makes it difficult for these modalities to image nanoscale inner cell structures and the distribution of internal cell components. Although super-resolution fluorescence microscopy, such as stimulated emission depletion microscopy (STED) and stochastic optical reconstruction microscopy (STORM), has successfully performed nanoscale biological imaging, these modalities require the use of exogenous fluorescence agents, which are unfavorable for biological samples. Our newly developed atomic force photoactivated microscopy (AFPM) can provide optical absorption images with nanoscale lateral resolution without any exogenous contrast agents. AFPM combines conventional atomic force microscopy (AFM) and an optical excitation system, and simultaneously provides multiple contrasts, such as the topography and magnitude of optical absorption. AFPM can detect the intrinsic optical absorption of samples with 8 nm lateral resolution, easily overcoming the diffraction limit. Using the label-free AFPM system, we have successfully imaged the optical absorption properties of a single melanoma cell (B16F10) and a rosette leaf epidermal cell of Arabidopsis (ecotype Columbia (Col-0)) with nanoscale lateral resolution. The remarkable images show the melanosome distribution of a melanoma cell and the biological structures of a plant cell. AFPM provides superior imaging of optical absorption with a nanoscale lateral resolution, and it promises to become widely used in biological and chemical research.
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Capillary absorption spectrometer and process for isotopic analysis of small samples
Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.; Moran, James J.; Newburn, Matthew K.; Blake, Thomas A.
2016-03-29
A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The method also permits sampling in vivo permitting real-time ambient studies of microbial communities.
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Capillary absorption spectrometer and process for isotopic analysis of small samples
DOE Office of Scientific and Technical Information (OSTI.GOV)
Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.
A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The process also permits sampling in vivo permitting real-time ambient studies of microbial communities.
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NASA Technical Reports Server (NTRS)
Borgstahl, Gloria (Inventor); Lovelace, Jeff (Inventor); Snell, Edward Holmes (Inventor); Bellamy, Henry (Inventor)
2008-01-01
The present invention provides a digital topography imaging system for determining the crystalline structure of a biological macromolecule, wherein the system employs a charge coupled device (CCD) camera with antiblooming circuitry to directly convert x-ray signals to electrical signals without the use of phosphor and measures reflection profiles from the x-ray emitting source after x-rays are passed through a sample. Methods for using said system are also provided.
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Compartmental genomics in living cells revealed by single-cell nanobiopsy.
Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader
2014-01-28
The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.
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Mougin, Christian; Artige, Emmanuelle; Marchand, Frédéric; Mondy, Samuel; Ratié, Céline; Sellier, Nadine; Castagnone-Sereno, Philippe; D'Acier, Armelle Cœur; Esmenjaud, Daniel; Faivre-Primot, Céline; Granjon, Laurent; Hamelet, Valérie; Lange, Frederic; Pagès, Sylvie; Rimet, Frédéric; Ris, Nicolas; Sallé, Guillaume
2018-04-19
The Biological Resource Centre for the Environment BRC4Env is a network of Biological Resource Centres (BRCs) and collections whose leading objectives are to improve the visibility of genetic and biological resources maintained by its BRCs and collections and to facilitate their use by a large research community, from agriculture research to life sciences and environmental sciences. Its added value relies on sharing skills, harmonizing practices, triggering projects in comparative biology, and ultimately proposing a single-entry portal to facilitate access to documented samples, taking into account the partnership policies of research institutions as well as the legal frame which varies with the biological nature of resources. BRC4Env currently includes three BRCs: the Centre for Soil Genetic Resources of the platform GenoSol, in partnership with the European Conservatory of Soil Samples; the Egg Parasitoids Collection (EP-Coll); and the collection of ichthyological samples, Colisa. BRC4Env is also associated to several biological collections: microbial consortia (entomopathogenic bacteria, freshwater microalgae…), terrestrial arthropods, nematodes (plant parasitic, entomopathogenic, animal parasitic...), and small mammals. The BRCs and collections of BRC4Env are involved in partnership with academic scientists, as well as private companies, in the fields of medicinal mining, biocontrol, sustainable agriculture, and additional sectors. Moreover, the staff of the BRCs is involved in many training courses for students from French licence degree to Ph.D, engineers, as well as ongoing training.
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NASA Astrophysics Data System (ADS)
Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.
2011-03-01
A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.
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From Animaculum to single molecules: 300 years of the light microscope.
Wollman, Adam J M; Nudd, Richard; Hedlund, Erik G; Leake, Mark C
2015-04-01
Although not laying claim to being the inventor of the light microscope, Antonj van Leeuwenhoek (1632-1723) was arguably the first person to bring this new technological wonder of the age properly to the attention of natural scientists interested in the study of living things (people we might now term 'biologists'). He was a Dutch draper with no formal scientific training. From using magnifying glasses to observe threads in cloth, he went on to develop over 500 simple single lens microscopes (Baker & Leeuwenhoek 1739 Phil. Trans. 41, 503-519. (doi:10.1098/rstl.1739.0085)) which he used to observe many different biological samples. He communicated his finding to the Royal Society in a series of letters (Leeuwenhoek 1800 The select works of Antony Van Leeuwenhoek, containing his microscopical discoveries in many of the works of nature, vol. 1) including the one republished in this edition of Open Biology. Our review here begins with the work of van Leeuwenhoek before summarizing the key developments over the last ca 300 years, which has seen the light microscope evolve from a simple single lens device of van Leeuwenhoek's day into an instrument capable of observing the dynamics of single biological molecules inside living cells, and to tracking every cell nucleus in the development of whole embryos and plants.
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We and others have shown that transition and maintenance of biological states is controlled by master regulator proteins, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined.
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Electrophoretic separator for purifying biologicals
NASA Technical Reports Server (NTRS)
1976-01-01
This technique separates a single narrow zone of sample mixture in an electrolyte medium into many zones containing a single component of the mixture and electrolyte between them. Since the densities of the separated zones generally differ from that of the intervening medium, such systems are gravitationally unstable and stabilization is required. The various techniques for stabilization include using the capillary space provided by thin films, the interstices of solid material such as filter paper and a variety of gel-forming substances.
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NASA Astrophysics Data System (ADS)
Qu, Jianan Y.; Sun, Qiqi
2017-02-01
The single or multi-photon microscopy based on fluorescent labelling and staining is a sensitive and quantitative method that is widely used in molecular biology and medical research for a variety of experimental, analytical, and quality control applications. However, label-free method is highly desirable in biology and medicine when performing long term live imaging of biological system and obtaining instant tissue examination during surgery procedures. Recently, our group found that femtosecond laser surgery turned a variety of biological tissues and protein samples into highly fluorescent substances. The newly formed fluorescent compounds produced during the laser surgery can be excited via single- and two-photon processes over broad wavelength ranges. We developed a combined confocal and two-photon spectroscopic microscope to characterize the fluorescence from the new compound systematically. The structures of the femtosecond laser treated tissue were studied using Raman spectroscopy and transmission electron microscopy. Our study revealed the mechanisms of the fluorescence emission form the new compound. Furthermore, we demonstrated the applications of the fluorescent compounds for instant evaluation of femtosecond laser microsurgery, study of stem cell responses to muscle injury and neuro-regeneration after spinal cord injury.
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Shui, Guanghou; Leong, Lai Peng
2005-02-23
In this study, a new approach was developed for screening and identifying antioxidants in biological samples. The approach was based on significant decreases of the intensities of ion peaks obtained from high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) upon reaction with 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals. HPLC-MS/MS was further applied to elucidate structures of antioxidant peaks characterized in a spiking test. The new approach could also be used to monitor the reactivity of antioxidants in biological sample with free radicals. The approach was successfully applied to the identification of antioxidants in salak (Salacca edulis Reinw), a tropical fruit that is reported to be a very good source of natural antioxidants, but it was still not clear which compounds were responsible for its antioxidant property. The antioxidants in salak were identified to be chlorogenic acid, (-)-epicatechin, and singly linked proanthocyanidins that mainly existed as dimers through hexamers of catechin or epicatechin. In salak, chlorogenic acid was identified to be an antioxidant of the slow reaction type as it reacted with free radicals much more slowly than either (-)-epicatechin or proanthocyanidins. The new approach was proved to be useful for the characterization and identification of antioxidants in biological samples as a mass detector combined with an HPLC separation system not only serves as an ideal tool to monitor free radical active components but also provides their possible chemical structures in a biological sample.
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Li, Chao; Yu, Jiaquan; Schehr, Jennifer; Berry, Scott M; Leal, Ticiana A; Lang, Joshua M; Beebe, David J
2018-05-23
The concept of high liquid repellency in multi-liquid-phase systems (e.g., aqueous droplets in an oil background) has been applied to areas of biomedical research to realize intrinsic advantages not available in single-liquid-phase systems. Such advantages have included minimizing analyte loss, facile manipulation of single-cell samples, elimination of biofouling, and ease of use regarding loading and retrieving of the sample. In this paper, we present generalized design rules for predicting the wettability of solid-liquid-liquid systems (especially for discrimination between exclusive liquid repellency (ELR) and finite liquid repellency) to extend the applications of ELR. We then apply ELR to two model systems with open microfluidic design in cell biology: (1) in situ underoil culture and combinatorial coculture of mammalian cells in order to demonstrate directed single-cell multiencapsulation with minimal waste of samples as compared to stochastic cell seeding and (2) isolation of a pure population of circulating tumor cells, which is required for certain downstream analyses including sequencing and gene expression profiling.
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Liu, Wen-Te; Bien, Mauo-Ying; Chuang, Kai-Jen; Chang, Ta-Yuan; Jones, Tim; BéruBé, Kelly; Lalev, Georgi; Tsai, Dai-Hua; Chuang, Hsiao-Chi; Cheng, Tsun-Jen
2014-09-15
To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.
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Khrustaleva, A M; Klovach, N V; Gritsenko, O F; Seeb, J E
2014-07-01
The variability of 45 single nucleotide polymorphism (SNP) loci was studied in nine samples of the sockeye salmon Oncorhynchus nerka from the rivers of southwestern Kamchatka. The Wahlund effect, gametic disequilibrium at some loci, and a decrease in interpopulation genetic diversity estimates observed in samples from the Bolshaya River outlet are explained in terms of the samples' heterogeneity. Partitioning of mixed samples using some biological characteristics of the individuals led to a noticeable decrease in the frequency of these phenomena. It was demonstrated that the allelic diversity between the populations within the river Plotnikovs accounted for the larger part of genetic variation, as compared to the differentiation between the basins. The SNP loci responsible for intra- and interpopulation differentiation of sockeye salmon from the rivers of southwestern Kamchatka were identified. Some recommendations for field population genetic studies of Asian sockeye salmon were formulated.
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Simulations of single-particle imaging of hydrated proteins with x-ray free-electron lasers
NASA Astrophysics Data System (ADS)
Fortmann-Grote, C.; Bielecki, J.; Jurek, Z.; Santra, R.; Ziaja-Motyka, B.; Mancuso, A. P.
2017-08-01
We employ start-to-end simulations to model coherent diffractive imaging of single biomolecules using x-ray free electron lasers. This technique is expected to yield new structural information about biologically relevant macromolecules thanks to the ability to study the isolated sample in its natural environment as opposed to crystallized or cryogenic samples. The effect of the solvent on the diffraction pattern and interpretability of the data is an open question. We present first results of calculations where the solvent is taken into account explicitly. They were performed with a molecular dynamics scheme for a sample consisting of a protein and a hydration layer of varying thickness. Through R-factor analysis of the simulated diffraction patterns from hydrated samples, we show that the scattering background from realistic hydration layers of up to 3 Å thickness presents no obstacle for the resolution of molecular structures at the sub-nm level.
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Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C.; Burnum-Johnson, Kristin E.; Kim, Young-Mo; Kyle, Jennifer E.; Matzke, Melissa M.; Shukla, Anil K.; Chu, Rosalie K.; Schepmoes, Athena A.; Jacobs, Jon M.; Baric, Ralph S.; Webb-Robertson, Bobbie-Jo; Smith, Richard D.
2016-01-01
ABSTRACT Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. The metabolite, protein, and lipid extraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro, and clinical). IMPORTANCE In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample. Author Video: An author video summary of this article is available. PMID:27822525
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Ackelsberg, Joel; Leykam, Frederic M; Hazi, Yair; Madsen, Larry C; West, Todd H; Faltesek, Anthony; Henderson, Gavin D; Henderson, Christopher L; Leighton, Terrance
2011-09-01
Native air sampling (NAS) is distinguished from dedicated air sampling (DAS) devices (eg, BioWatch) that are deployed to detect aerosol disseminations of biological threat agents. NAS uses filter samples from heating, ventilation, and air conditioning (HVAC) systems in commercial properties for environmental sampling after DAS detection of biological threat agent incidents. It represents an untapped, scientifically sound, efficient, widely distributed, and comparably inexpensive resource for postevent environmental sampling. Calculations predict that postevent NAS would be more efficient than environmental surface sampling by orders of magnitude. HVAC filter samples could be collected from pre-identified surrounding NAS facilities to corroborate the DAS alarm and delineate the path taken by the bioaerosol plume. The New York City (NYC) Native Air Sampling Pilot Project explored whether native air sampling would be acceptable to private sector stakeholders and could be implemented successfully in NYC. Building trade associations facilitated outreach to and discussions with property owners and managers, who expedited contact with building managers of candidate NAS properties that they managed or owned. Nominal NAS building requirements were determined; procedures to identify and evaluate candidate NAS facilities were developed; data collection tools and other resources were designed and used to expedite candidate NAS building selection and evaluation in Manhattan; and exemplar environmental sampling playbooks for emergency responders were completed. In this sample, modern buildings with single or few corporate tenants were the best NAS candidate facilities. The Pilot Project successfully demonstrated that in one urban setting a native air sampling strategy could be implemented with effective public-private collaboration.
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Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics.
Farlik, Matthias; Sheffield, Nathan C; Nuzzo, Angelo; Datlinger, Paul; Schönegger, Andreas; Klughammer, Johanna; Bock, Christoph
2015-03-03
Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Fully Integrated Microfluidic Device for Direct Sample-to-Answer Genetic Analysis
NASA Astrophysics Data System (ADS)
Liu, Robin H.; Grodzinski, Piotr
Integration of microfluidics technology with DNA microarrays enables building complete sample-to-answer systems that are useful in many applications such as clinic diagnostics. In this chapter, a fully integrated microfluidic device [1] that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and a DNA microarray sensor to perform DNA analysis of complex biological sample solutions is present. This device can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization, and electrochemical detection. A few novel microfluidic techniques were developed and employed. A micromix-ing technique based on a cavitation microstreaming principle was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads. This technique was also employed to accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from ~mL whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.
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NASA Astrophysics Data System (ADS)
Preibisch, Stephan; Rohlfing, Torsten; Hasak, Michael P.; Tomancak, Pavel
2008-03-01
Single Plane Illumination Microscopy (SPIM; Huisken et al., Nature 305(5686):1007-1009, 2004) is an emerging microscopic technique that enables live imaging of large biological specimens in their entirety. By imaging the living biological sample from multiple angles SPIM has the potential to achieve isotropic resolution throughout even relatively large biological specimens. For every angle, however, only a relatively shallow section of the specimen is imaged with high resolution, whereas deeper regions appear increasingly blurred. In order to produce a single, uniformly high resolution image, we propose here an image mosaicing algorithm that combines state of the art groupwise image registration for alignment with content-based image fusion to prevent degrading of the fused image due to regional blurring of the input images. For the registration stage, we introduce an application-specific groupwise transformation model that incorporates per-image as well as groupwise transformation parameters. We also propose a new fusion algorithm based on Gaussian filters, which is substantially faster than fusion based on local image entropy. We demonstrate the performance of our mosaicing method on data acquired from living embryos of the fruit fly, Drosophila, using four and eight angle acquisitions.
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Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles
NASA Astrophysics Data System (ADS)
Tseng, Peter
Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to generate user-controllable (time-varying and localizable), massively parallel forces on arrays of cells mediated by coalesced ensembles of magnetic nanoparticles. The above process is simplified and adapted for single cell analysis by precisely aligning fibronectin patterned cells to a single flanking micromagnet. The cells are loaded with magnetic-fluorescent nanoparticles, which are then localized to uniform positions at the internal edge of the cell membrane over huge arrays of cells using large external fields, allowing us to conduct composed studies on cellular response to force. By applying forces approaching the yield tension (5 nN / mum) of single cells, we are able to generate highly coordinated responses in cellular behavior. We discover that increasing tension generates highly directed, PAK-dependent leading-edge type filopodia that increase in intensity with rising tension. In addition, we find that our generated forces can simulate cues created during cellular mitosis, as we are consistently able to generate significant (45 to 90 degree) biasing of the metaphase plate during cell division. Large sample size and rapid sample generation also allow us to analyze cells at an unprecedented rate---a single sample can simultaneously stimulate thousands of cells for high statistical accuracy in measurements. We believe these approaches have potential not just as a tool to study single-cell response, but as a means of cell control, potentially through modifying cell movement, division, or differentiation. More generally, once approaches to release nanoparticles from endosomes are implemented, the technique provides a platform to dynamically apply a range of localized stimuli arbitrarily within cells. Through the bioconjugation of proteins, nucleic acids, small molecules, or whole organelles a broad range of questions should be accessible concerning molecular localization and its importance in cell function.
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Development of a Bioaerosol single particle detector (BIO IN) for the Fast Ice Nucleus CHamber FINCH
NASA Astrophysics Data System (ADS)
Bundke, U.; Reimann, B.; Nillius, B.; Jaenicke, R.; Bingemer, H.
2010-02-01
In this work we present the setup and first tests of our new BIO IN detector. This detector was constructed to classify atmospheric ice nuclei (IN) for their biological content. It is designed to be coupled to the Fast Ice Nucleus CHamber FINCH. If one particle acts as an ice nucleus, it will be at least partly covered with ice at the end of the development section of the FINCH chamber. The device combines an auto-fluorescence detector and a circular depolarization detector for simultaneous detection of biological material and discrimination between water droplets, ice crystals and non activated large aerosol particles. The excitation of biological material with UV light and analysis of auto-fluorescence is a common principle used for flow cytometry, fluorescence microscopy, spectroscopy and imaging. The detection of auto-fluorescence of airborne single particles demands some more experimental effort. However, expensive commercial sensors are available for special purposes, e.g. size distribution measurements. But these sensors will not fit the specifications needed for the FINCH IN counter (e.g. high sample flow of up 10 LPM). The newly developed -low cost- BIO IN sensor uses a single high-power UV LED for the electronic excitation instead of much more expensive UV lasers. Other key advantages of the new sensor are the low weight, compact size, and the little effect on the aerosol sample, which allows it to be coupled with other instruments for further analysis. The instrument will be flown on one of the first missions of the new German research aircraft "HALO" (High Altitude and LOng range).
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Simultaneous extraction of proteins and metabolites from cells in culture
Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten
2014-01-01
Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938
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Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy
Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader
2014-01-01
The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711
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Live cell imaging at the Munich ion microbeam SNAKE - a status report.
Drexler, Guido A; Siebenwirth, Christian; Drexler, Sophie E; Girst, Stefanie; Greubel, Christoph; Dollinger, Günther; Friedl, Anna A
2015-02-18
Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.
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Integrated Raman and angular scattering of single biological cells
NASA Astrophysics Data System (ADS)
Smith, Zachary J.
2009-12-01
Raman, or inelastic, scattering and angle-resolved elastic scattering are two optical processes that have found wide use in the study of biological systems. Raman scattering quantitatively reports on the chemical composition of a sample by probing molecular vibrations, while elastic scattering reports on the morphology of a sample by detecting structure-induced coherent interference between incident and scattered light. We present the construction of a multimodal microscope platform capable of gathering both elastically and inelastically scattered light from a 38 mum2 region in both epi- and trans-illumination geometries. Simultaneous monitoring of elastic and inelastic scattering from a microscopic region allows noninvasive characterization of a living sample without the need for exogenous dyes or labels. A sample is illuminated either from above or below with a focused 785 nm TEM00 mode laser beam, with elastic and inelastic scattering collected by two separate measurement arms. The measurements may be made either simultaneously, if identical illumination geometries are used, or sequentially, if the two modalities utilize opposing illumination paths. In the inelastic arm, Stokes-shifted light is dispersed by a spectrograph onto a CCD array. In the elastic scattering collection arm, a relay system images the microscope's back aperture onto a CCD detector array to yield an angle-resolved elastic scattering pattern. Post-processing of the inelastic scattering to remove fluorescence signals yields high quality Raman spectra that report on the sample's chemical makeup. Comparison of the elastically scattered pupil images to generalized Lorenz-Mie theory yields estimated size distributions of scatterers within the sample. In this thesis we will present validations of the IRAM instrument through measurements performed on single beads of a few microns in size, as well as on ensembles of sub-micron particles of known size distributions. The benefits and drawbacks of the epi- and trans-illumination modalities are also discussed. In addition, transilluminated Raman and elastic-scattering spectra were obtained from several biological test-cases, including Streptococcus pneumoniae, baker's yeast, and single human immune cells. Both the Raman and elastic-scattering channels extract information from these samples that are well in line with their known characteristics from the literature. Finally, we report on an experiment in which CD8+ T lymphocytes were stimulated by exposure to the antigens staphylococcal enterotoxin B and phorbol myristate acetate. Clear chemical and morphological differences were observed between the activated and unactivated cells, with the results correlating well to analysis performed on parallel samples using fluorescent stains and a flow cytometer.
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On-chip Magnetic Separation and Cell Encapsulation in Droplets
NASA Astrophysics Data System (ADS)
Chen, A.; Byvank, T.; Bharde, A.; Miller, B. L.; Chalmers, J. J.; Sooryakumar, R.; Chang, W.-J.; Bashir, R.
2012-02-01
The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment could prevent cross-contamination, provide high recovery yield and ability to study biological traits at a single cell level These advantages of on-chip biological experiments contrast to conventional methods, which require bulk samples that provide only averaged information on cell metabolism. We report on a device that integrates microfluidic technology with a magnetic tweezers array to combine the functionality of separation and encapsulation of objects such as immunomagnetically labeled cells or magnetic beads into pico-liter droplets on the same chip. The ability to control the separation throughput that is independent of the hydrodynamic droplet generation rate allows the encapsulation efficiency to be optimized. The device can potentially be integrated with on-chip labeling and/or bio-detection to become a powerful single-cell analysis device.
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Lazzarino, Giacomo; Longo, Salvatore; Amorini, Angela Maria; Di Pietro, Valentina; D'Urso, Serafina; Lazzarino, Giuseppe; Belli, Antonio; Tavazzi, Barbara
2017-12-08
Fat-soluble vitamins and antioxidants are of relevance in health and disease. Current methods to extract these compounds from biological fluids mainly need use of multi-steps and multi organic solvents. They are time-consuming and difficult to apply to treat simultaneously large sample number. We here describe a single-step, one solvent extraction of fat-soluble vitamins and antioxidants from biological fluids, and the chromatographic separation of all-trans-retinoic acid, 25-hydroxycholecalciferol, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8'-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, phylloquinone, lycopene, α-carotene, β-carotene and coenzyme Q 10 . Extraction is obtained by adding one volume of biological fluid to two acetonitrile volumes, vortexing for 60s and incubating for 60min at 37°C under agitation. HPLC separation occurs in 30min using Hypersil C18, 100×4.6mm, 5μm particle size column, gradient from 70% methanol+30% H 2 O to 100% acetonitrile, flow rate of 1.0ml/min and 37°C column temperature. Compounds are revealed using highly sensitive UV-VIS diode array detector. The HPLC method suitability was assessed in terms of sensitivity, reproducibility and recovery. Using the present extraction and chromatographic conditions we obtained values of the fat-soluble vitamins and antioxidants in serum from 50 healthy controls similar to those found in literature. Additionally, the profile of these compounds was also measured in seminal plasma from 20 healthy fertile donors. Results indicate that this simple, rapid and low cost sample processing is suitable to extract fat-soluble vitamins and antioxidants from biological fluids and can be applied in clinical and nutritional studies. Copyright © 2017 Elsevier B.V. All rights reserved.
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Nanoscaled aptasensors for multi-analyte sensing
Saberian-Borujeni, Mehdi; Johari-Ahar, Mohammad; Hamzeiy, Hossein; Barar, Jaleh; Omidi, Yadollah
2014-01-01
Introduction: Nanoscaled aptamers (Aps), as short single-stranded DNA or RNA oligonucleotides, are able to bind to their specific targets with high affinity, upon which they are considered as powerful diagnostic and analytical sensing tools (the so-called "aptasensors"). Aptamers are selected from a random pool of oligonucleotides through a procedure known as "systematic evolution of ligands by exponential enrichment". Methods: In this work, the most recent studies in the field of aptasensors are reviewed and discussed with a main focus on the potential of aptasensors for the multianalyte detection(s). Results: Due to the specific folding capability of aptamers in the presence of analyte, aptasensors have substantially successfully been exploited for the detection of a wide range of small and large molecules (e.g., drugs and their metabolites, toxins, and associated biomarkers in various diseases) at very low concentrations in the biological fluids/samples even in presence of interfering species. Conclusion: Biological samples are generally considered as complexes in the real biological media. Hence, the development of aptasensors with capability to determine various targets simultaneously within a biological matrix seems to be our main challenge. To this end, integration of various key scientific dominions such as bioengineering and systems biology with biomedical researches are inevitable. PMID:25671177
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Genetic variation in avocado stem weevils Copturus aguacatae (Coleoptera: Curculionidae) in Mexico.
Engstrand, Rachel C; Cibrián Tovar, Juan; Cibrián-Jaramillo, Angélica; Kolokotronis, Sergios-Orestis
2010-12-01
The avocado stem weevil Copturus aguacatae is an important pest in avocado plantations. Its presence hinders the production and marketing of avocado in Mexico, the largest avocado producer worldwide. Biological control through pheromone synthesis, a strategy favored over chemical control in crops, is currently limited by difficult field identification of this species. Using DNA barcoding, we examine the patterns of genetic variation of C. aguacatae in avocado trees in Mexico to help facilitate its identification and biological control. We show that there is one single species of avocado stem weevil throughout the sampled sites in Mexico. Overall, haplotype diversity is high, with Oaxaca forming one distinct group and all other sampled populations are admixed irrespective of geographic origin. The results suggest that high gene flow is maintained in this species and that a global strategy for biocontrol can be designed and implemented throughout the sampled range.
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Single cell genomic quantification by non-fluorescence nonlinear microscopy
NASA Astrophysics Data System (ADS)
Kota, Divya; Liu, Jing
2017-02-01
Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.
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From Animaculum to single molecules: 300 years of the light microscope
Wollman, Adam J. M.; Nudd, Richard; Hedlund, Erik G.; Leake, Mark C.
2015-01-01
Although not laying claim to being the inventor of the light microscope, Antonj van Leeuwenhoek (1632–1723) was arguably the first person to bring this new technological wonder of the age properly to the attention of natural scientists interested in the study of living things (people we might now term ‘biologists’). He was a Dutch draper with no formal scientific training. From using magnifying glasses to observe threads in cloth, he went on to develop over 500 simple single lens microscopes (Baker & Leeuwenhoek 1739 Phil. Trans. 41, 503–519. (doi:10.1098/rstl.1739.0085)) which he used to observe many different biological samples. He communicated his finding to the Royal Society in a series of letters (Leeuwenhoek 1800 The select works of Antony Van Leeuwenhoek, containing his microscopical discoveries in many of the works of nature, vol. 1) including the one republished in this edition of Open Biology. Our review here begins with the work of van Leeuwenhoek before summarizing the key developments over the last ca 300 years, which has seen the light microscope evolve from a simple single lens device of van Leeuwenhoek's day into an instrument capable of observing the dynamics of single biological molecules inside living cells, and to tracking every cell nucleus in the development of whole embryos and plants. PMID:25924631
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NASA Astrophysics Data System (ADS)
Cizdziel, James V.; Tolbert, Candice; Brown, Garry
2010-02-01
A Direct Mercury Analyzer (DMA) based on sample combustion, concentration of mercury by amalgamation with gold, and cold vapor atomic absorption spectrometry (CVAAS) was coupled to a mercury-specific cold vapor atomic fluorescence spectrometer (CVAFS). The purpose was to evaluate combustion-AFS, a technique which is not commercially available, for low-level analysis of mercury in environmental and biological samples. The experimental setup allowed for comparison of dual measurements of mercury (AAS followed by AFS) for a single combustion event. The AFS instrument control program was modified to properly time capture of mercury from the DMA, avoiding deleterious combustion products from reaching its gold traps. Calibration was carried out using both aqueous solutions and solid reference materials. The absolute detection limits for mercury were 0.002 ng for AFS and 0.016 ng for AAS. Recoveries for reference materials ranged from 89% to 111%, and the precision was generally found to be <10% relative standard deviation (RSD). The two methods produced similar results for samples of hair, finger nails, coal, soil, leaves and food stuffs. However, for samples with mercury near the AAS detection limit (e.g., filter paper spotted with whole blood and segments of tree rings) the signal was still quantifiable with AFS, demonstrating the lower detection limit and greater sensitivity of AFS. This study shows that combustion-AFS is feasible for the direct analysis of low levels of mercury in solid samples that would otherwise require time-consuming and contamination-prone digestion.
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NASA Astrophysics Data System (ADS)
Nedosekin, Dmitry A.; Nolan, Jacqueline; Biris, Alexandru S.; Zharov, Vladimir P.
2017-03-01
Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in collaboration with other Arkansas Universities and the FDA-based National Center of Toxicological Research in Jefferson, AR is developing novel techniques for rapid quantification of graphene-based nanomaterials (GBNs) in various biological samples. All-carbon GBNs have wide range of potential applications in industry, agriculture, food processing and medicine; however, quantification of GBNs is difficult in carbon reach biological tissues. The accurate quantification of GBNs is essential for research on material toxicity and the development of GBNs-based drug delivery platforms. We have developed microscopy and cytometry platforms for detection and quantification of GBNs in single cells, tissue and blood samples using photoacoustic contrast of GBNs. We demonstrated PA quantification of individual graphene uptake by single cells. High-resolution PA microscopy provided mapping of GBN distribution within live cells to establish correlation with intracellular toxic phenomena using apoptotic and necrotic assays. This new methodology and corresponding technical platform provide the insight on possible toxicological risks of GBNs at singe cells levels. In addition, in vivo PA image flow cytometry demonstrated the capability to monitor of GBNs pharmacokinetics in mouse model and to map the resulting biodistribution of GBNs in mouse tissues. The integrated PA platform provided an unprecedented sensitivity toward GBNs and allowed to enhance conventional toxicology research by providing a direct correlation between uptake of GBNs at a single cell level and cell viability status.
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Nucleic acid purification from plants, animals and microbes in under 30 seconds
Zou, Yiping; Wang, Yuling; Wee, Eugene; Turni, Conny; Blackall, Patrick J.; Trau, Matt; Botella, Jose Ramon
2017-01-01
Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries. PMID:29161268
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The olfactory hole-board test in rats: a new paradigm to study aversion and preferences to odors
Wernecke, Kerstin E. A.; Fendt, Markus
2015-01-01
Odors of biological relevance (e.g., predator odors, sex odors) are known to effectively influence basic survival needs of rodents such as anti-predatory defensiveness and mating behaviors. Research focused on the effects of these odors on rats’ behavior mostly includes multi-trial paradigms where animals experience single odor exposures in subsequent, separated experimental sessions. In the present study, we introduce a modification of the olfactory hole-board test that allows studying the effects of different odors on rats’ behavior within single trials. First, we demonstrated that the corner holes of the hole-board were preferentially visited by rats. The placement of different odors under the corner holes changed this hole preference. We showed that holes with carnivore urine samples were avoided, while corner holes with female rat urine samples were preferred. Furthermore, corner holes with urine samples from a carnivore, herbivore, and omnivore were differentially visited indicating that rats can discriminate these odors. To test whether anxiolytic treatment specifically modulates the avoidance of carnivore urine holes, we treated rats with buspirone. Buspirone treatment completely abolished the avoidance of carnivore urine holes. Taken together, our findings indicate that the olfactory hole-board test is a valuable tool for measuring avoidance and preference responses to biologically relevant odors. PMID:26379516
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Yu, Feiqiao Brian; Blainey, Paul C; Schulz, Frederik; Woyke, Tanja; Horowitz, Mark A; Quake, Stephen R
2017-07-05
Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.
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NASA Astrophysics Data System (ADS)
Tinne, N.; Matthias, B.; Kranert, F.; Wetzel, C.; Krüger, A.; Ripken, T.
2016-03-01
The interaction effect of photodisruption, which is used for dissection of biological tissue with fs-laser pulses, has been intensively studied inside water as prevalent sample medium. In this case, the single effect is highly reproducible and, hence, the method of time-resolved photography is sufficiently applicable. In contrast, the reproducibility significantly decreases analyzing more solid and anisotropic media like biological tissue. Therefore, a high-speed photographic approach is necessary in this case. The presented study introduces a novel technique for high-speed photography based on the principle of chromatic encoding. For illumination of the region of interest within the sample medium, the light paths of up to 12 LEDs with various emission wavelengths are overlaid via optical filters. Here, MOSFET-electronics provide a LED flash with a duration <100 ns; the diodes are externally triggered with a distinct delay for every LED. Furthermore, the different illumination wavelengths are chromatically separated again for detection via camera chip. Thus, the experimental setup enables the generation of a time-sequence of <= 12 images of a single cavitation bubble dynamics. In comparison to conventional time-resolved photography, images in sample media like water and HEMA show the significant advantages of this novel illumination technique. In conclusion, the results of this study are of great importance for the fundamental evaluation of the laser-tissue interaction inside anisotropic biological tissue and for the optimization of the surgical process with high-repetition rate fs-lasers. Additionally, this application is also suitable for the investigation of other microscopic, ultra-fast events in transparent inhomogeneous materials.
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Single grating x-ray imaging for dynamic biological systems
NASA Astrophysics Data System (ADS)
Morgan, Kaye S.; Paganin, David M.; Parsons, David W.; Donnelley, Martin; Yagi, Naoto; Uesugi, Kentaro; Suzuki, Yoshio; Takeuchi, Akihisa; Siu, Karen K. W.
2012-07-01
Biomedical studies are already benefiting from the excellent contrast offered by phase contrast x-ray imaging, but live imaging work presents several challenges. Living samples make it particularly difficult to achieve high resolution, sensitive phase contrast images, as exposures must be short and cannot be repeated. We therefore present a single-exposure, high-flux method of differential phase contrast imaging [1, 2, 3] in the context of imaging live airways for Cystic Fibrosis (CF) treatment assessment [4]. The CF study seeks to non-invasively observe the liquid lining the airways, which should increase in depth in response to effective treatments. Both high spatial resolution and sensitivity are required in order to track micron size changes in a liquid that is not easily differentiated from the tissue on which it lies. Our imaging method achieves these goals by using a single attenuation grating or grid as a reference pattern, and analyzing how the sample deforms the pattern to quantitatively retrieve the phase depth of the sample. The deformations are mapped at each pixel in the image using local cross-correlations comparing each 'sample and pattern' image with a reference 'pattern only' image taken before the sample is introduced. This produces a differential phase image, which may be integrated to give the sample phase depth.
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NASA Astrophysics Data System (ADS)
Zoriniants, George; Masia, Francesco; Giannakopoulou, Naya; Langbein, Wolfgang; Borri, Paola
2017-10-01
Single nanoparticle tracking using optical microscopy is a powerful technique with many applications in biology, chemistry, and material sciences. Despite significant advances, localizing objects with nanometric position precision in a scattering environment remains challenging. Applied methods to achieve contrast are dominantly fluorescence based, with fundamental limits in the emitted photon fluxes arising from the excited-state lifetime as well as photobleaching. Here, we show a new four-wave-mixing interferometry technique, whereby the position of a single nonfluorescing gold nanoparticle of 25-nm radius is determined with 16 nm precision in plane and 3 nm axially from rapid single-point measurements at 1-ms acquisition time by exploiting optical vortices. The precision in plane is consistent with the photon shot-noise, while axially it is limited by the nano-positioning sample stage, with an estimated photon shot-noise limit of 0.5 nm. The detection is background-free even inside biological cells. The technique is also uniquely sensitive to particle asymmetries of only 0.5% ellipticity, corresponding to a single atomic layer of gold, as well as particle orientation. This method opens new ways of unraveling single-particle trafficking within complex 3D architectures.
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Jeong, Heon-Ho; Lee, Byungjin; Jin, Si Hyung; Jeong, Seong-Geun; Lee, Chang-Soo
2016-04-26
Droplet-based microfluidics enabling exquisite liquid-handling has been developed for diagnosis, drug discovery and quantitative biology. Compartmentalization of samples into a large number of tiny droplets is a great approach to perform multiplex assays and to improve reliability and accuracy using a limited volume of samples. Despite significant advances in microfluidic technology, individual droplet handling in pico-volume resolution is still a challenge in obtaining more efficient and varying multiplex assays. We present a highly addressable static droplet array (SDA) enabling individual digital manipulation of a single droplet using a microvalve system. In a conventional single-layer microvalve system, the number of microvalves required is dictated by the number of operation objects; thus, individual trap-and-release on a large-scale 2D array format is highly challenging. By integrating double-layer microvalves, we achieve a "balloon" valve that preserves the pressure-on state under released pressure; this valve can allow the selective releasing and trapping of 7200 multiplexed pico-droplets using only 1 μL of sample without volume loss. This selectivity and addressability completely arranged only single-cell encapsulated droplets from a mixture of droplet compositions via repetitive selective trapping and releasing. Thus, it will be useful for efficient handling of miniscule volumes of rare or clinical samples in multiplex or combinatory assays, and the selective collection of samples.
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B, Vinoth; Lai, Xin-Ji; Lin, Yu-Chih; Tu, Han-Yen; Cheng, Chau-Jern
2018-04-13
Digital holographic microtomography is a promising technique for three-dimensional (3D) measurement of the refractive index (RI) profiles of biological specimens. Measurement of the RI distribution of a free-floating single living cell with an isotropic superresolution had not previously been accomplished. To the best of our knowledge, this is the first study focusing on the development of an integrated dual-tomographic (IDT) imaging system for RI measurement of an unlabelled free-floating single living cell with an isotropic superresolution by combining the spatial frequencies of full-angle specimen rotation with those of beam rotation. A novel 'UFO' (unidentified flying object) like shaped coherent transfer function is obtained. The IDT imaging system does not require any complex image-processing algorithm for 3D reconstruction. The working principle was successfully demonstrated and a 3D RI profile of a single living cell, Candida rugosa, was obtained with an isotropic superresolution. This technology is expected to set a benchmark for free-floating single live sample measurements without labeling or any special sample preparations for the experiments.
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Using Genotype Abundance to Improve Phylogenetic Inference
Mesin, Luka; Victora, Gabriel D; Minin, Vladimir N; Matsen, Frederick A
2018-01-01
Abstract Modern biological techniques enable very dense genetic sampling of unfolding evolutionary histories, and thus frequently sample some genotypes multiple times. This motivates strategies to incorporate genotype abundance information in phylogenetic inference. In this article, we synthesize a stochastic process model with standard sequence-based phylogenetic optimality, and show that tree estimation is substantially improved by doing so. Our method is validated with extensive simulations and an experimental single-cell lineage tracing study of germinal center B cell receptor affinity maturation. PMID:29474671
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2007-09-01
limitations due to so-called "bottle effects" produced by confining production to a single bottle, eliminating grazers, trace metal contamination from the...1, b - 2/3) (Levich, 1962 ) or can be determined by modeling studies of characteristic bubble populations (a = 0.7, b = 0.35) (Keeling, 1993). In this...artifacts associated with the early sampling method. In addition, some of the samples with large supersaturations may have been contaminated with
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Raman sorting and identification of single living micro-organisms with optical tweezers
NASA Astrophysics Data System (ADS)
Xie, Changan; Chen, De; Li, Yong-Qing
2005-07-01
We report on a novel technique for sorting and identification of single biological cells and food-borne bacteria based on laser tweezers and Raman spectroscopy (LTRS). With this technique, biological cells of different physiological states in a sample chamber were identified by their Raman spectral signatures and then they were selectively manipulated into a clean collection chamber with optical tweezers through a microchannel. As an example, we sorted the live and dead yeast cells into the collection chamber and validated this with a standard staining technique. We also demonstrated that bacteria existing in spoiled foods could be discriminated from a variety of food particles based on their characteristic Raman spectra and then isolated with laser manipulation. This label-free LTRS sorting technique may find broad applications in microbiology and rapid examination of food-borne diseases.
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Electrochemical imaging of cells and tissues
Lin, Tzu-En; Rapino, Stefania; Girault, Hubert H.
2018-01-01
The technological and experimental progress in electrochemical imaging of biological specimens is discussed with a view on potential applications for skin cancer diagnostics, reproductive medicine and microbial testing. The electrochemical analysis of single cell activity inside cell cultures, 3D cellular aggregates and microtissues is based on the selective detection of electroactive species involved in biological functions. Electrochemical imaging strategies, based on nano/micrometric probes scanning over the sample and sensor array chips, respectively, can be made sensitive and selective without being affected by optical interference as many other microscopy techniques. The recent developments in microfabrication, electronics and cell culturing/tissue engineering have evolved in affordable and fast-sampling electrochemical imaging platforms. We believe that the topics discussed herein demonstrate the applicability of electrochemical imaging devices in many areas related to cellular functions. PMID:29899947
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Scalable metagenomic taxonomy classification using a reference genome database
Ames, Sasha K.; Hysom, David A.; Gardner, Shea N.; Lloyd, G. Scott; Gokhale, Maya B.; Allen, Jonathan E.
2013-01-01
Motivation: Deep metagenomic sequencing of biological samples has the potential to recover otherwise difficult-to-detect microorganisms and accurately characterize biological samples with limited prior knowledge of sample contents. Existing metagenomic taxonomic classification algorithms, however, do not scale well to analyze large metagenomic datasets, and balancing classification accuracy with computational efficiency presents a fundamental challenge. Results: A method is presented to shift computational costs to an off-line computation by creating a taxonomy/genome index that supports scalable metagenomic classification. Scalable performance is demonstrated on real and simulated data to show accurate classification in the presence of novel organisms on samples that include viruses, prokaryotes, fungi and protists. Taxonomic classification of the previously published 150 giga-base Tyrolean Iceman dataset was found to take <20 h on a single node 40 core large memory machine and provide new insights on the metagenomic contents of the sample. Availability: Software was implemented in C++ and is freely available at http://sourceforge.net/projects/lmat Contact: allen99@llnl.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23828782
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Discovery of 20,000 RAD-SNPs and development of a 52-SNP array for monitoring river otters
Jeffrey B. Stetz; Seth Smith; Michael A. Sawaya; Alan B. Ramsey; Stephen J. Amish; Michael K. Schwartz; Gordon Luikart
2016-01-01
Many North American river otter (Lontra canadensis) populations are threatened or recovering but are difficult to study because they occur at low densities, it is difficult to visually identify individuals, and they inhabit aquatic environments that accelerate degradation of biological samples. Single nucleotide polymorphisms (SNPs) can improve our ability to...
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Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation.
Dueck, Hannah; Khaladkar, Mugdha; Kim, Tae Kyung; Spaethling, Jennifer M; Francis, Chantal; Suresh, Sangita; Fisher, Stephen A; Seale, Patrick; Beck, Sheryl G; Bartfai, Tamas; Kuhn, Bernhard; Eberwine, James; Kim, Junhyong
2015-06-09
Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.
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Fraley, Stephanie I; Hardick, Justin; Masek, Billie J; Jo Masek, Billie; Athamanolap, Pornpat; Rothman, Richard E; Gaydos, Charlotte A; Carroll, Karen C; Wakefield, Teresa; Wang, Tza-Huei; Yang, Samuel
2013-10-01
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.
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Advances and applications of occupancy models
Bailey, Larissa; MacKenzie, Darry I.; Nichols, James D.
2013-01-01
Summary: The past decade has seen an explosion in the development and application of models aimed at estimating species occurrence and occupancy dynamics while accounting for possible non-detection or species misidentification. We discuss some recent occupancy estimation methods and the biological systems that motivated their development. Collectively, these models offer tremendous flexibility, but simultaneously place added demands on the investigator. Unlike many mark–recapture scenarios, investigators utilizing occupancy models have the ability, and responsibility, to define their sample units (i.e. sites), replicate sampling occasions, time period over which species occurrence is assumed to be static and even the criteria that constitute ‘detection’ of a target species. Subsequent biological inference and interpretation of model parameters depend on these definitions and the ability to meet model assumptions. We demonstrate the relevance of these definitions by highlighting applications from a single biological system (an amphibian–pathogen system) and discuss situations where the use of occupancy models has been criticized. Finally, we use these applications to suggest future research and model development.
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Little, J P; Tevelen, G; Adam, C J; Evans, J H; Pearcy, M J
2009-07-01
Biological tissues are subjected to complex loading states in vivo and in order to define constitutive equations that effectively simulate their mechanical behaviour under these loads, it is necessary to obtain data on the tissue's response to multiaxial loading. Single axis and shear testing of biological tissues is often carried out, but biaxial testing is less common. We sought to design and commission a biaxial compression testing device, capable of obtaining repeatable data for biological samples. The apparatus comprised a sealed stainless steel pressure vessel specifically designed such that a state of hydrostatic compression could be created on the test specimen while simultaneously unloading the sample along one axis with an equilibrating tensile pressure. Thus a state of equibiaxial compression was created perpendicular to the long axis of a rectangular sample. For the purpose of calibration and commissioning of the vessel, rectangular samples of closed cell ethylene vinyl acetate (EVA) foam were tested. Each sample was subjected to repeated loading, and nine separate biaxial experiments were carried out to a maximum pressure of 204 kPa (30 psi), with a relaxation time of two hours between them. Calibration testing demonstrated the force applied to the samples had a maximum error of 0.026 N (0.423% of maximum applied force). Under repeated loading, the foam sample demonstrated lower stiffness during the first load cycle. Following this cycle, an increased stiffness, repeatable response was observed with successive loading. While the experimental protocol was developed for EVA foam, preliminary results on this material suggest that this device may be capable of providing test data for biological tissue samples. The load response of the foam was characteristic of closed cell foams, with consolidation during the early loading cycles, then a repeatable load-displacement response upon repeated loading. The repeatability of the test results demonstrated the ability of the test device to provide reproducible test data and the low experimental error in the force demonstrated the reliability of the test data.
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Approaches to Quality Risk Management When Using Single-Use Systems in the Manufacture of Biologics.
Ishii-Watabe, Akiko; Hirose, Akihiko; Katori, Noriko; Hashii, Norikata; Arai, Susumu; Awatsu, Hirotoshi; Eiza, Akira; Hara, Yoshiaki; Hattori, Hideshi; Inoue, Tomomi; Isono, Tetsuya; Iwakura, Masahiro; Kajihara, Daisuke; Kasahara, Nobuo; Matsuda, Hiroyuki; Murakami, Sei; Nakagawa, Taishiro; Okumura, Takehiro; Omasa, Takeshi; Takuma, Shinya; Terashima, Iyo; Tsukahara, Masayoshi; Tsutsui, Maiko; Yano, Takahiro; Kawasaki, Nana
2015-10-01
Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.
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Detection of Single Molecules Illuminated by a Light-Emitting Diode
Gerhardt, Ilja; Mai, Lijian; Lamas-Linares, Antía; Kurtsiefer, Christian
2011-01-01
Optical detection and spectroscopy of single molecules has become an indispensable tool in biological imaging and sensing. Its success is based on fluorescence of organic dye molecules under carefully engineered laser illumination. In this paper we demonstrate optical detection of single molecules on a wide-field microscope with an illumination based on a commercially available, green light-emitting diode. The results are directly compared with laser illumination in the same experimental configuration. The setup and the limiting factors, such as light transfer to the sample, spectral filtering and the resulting signal-to-noise ratio are discussed. A theoretical and an experimental approach to estimate these parameters are presented. The results can be adapted to other single emitter and illumination schemes. PMID:22346610
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Dowall, Stuart D; Graham, Victoria A; Tipton, Thomas R W; Hewson, Roger
2009-08-31
Work with highly pathogenic material mandates the use of biological containment facilities, involving microbiological safety cabinets and specialist laboratory engineering structures typified by containment level 3 (CL3) and CL4 laboratories. Consequences of working in high containment are the practical difficulties associated with containing specialist assays and equipment often essential for experimental analyses. In an era of increased interest in biodefence pathogens and emerging diseases, immunological analysis has developed rapidly alongside traditional techniques in virology and molecular biology. For example, in order to maximise the use of small sample volumes, multiplexing has become a more popular and widespread approach to quantify multiple analytes simultaneously, such as cytokines and chemokines. The luminex microsphere system allows for the detection of many cytokines and chemokines in a single sample, but the detection method of using aligned lasers and fluidics means that samples often have to be analysed in low containment facilities. In order to perform cytokine analysis in materials from high containment (CL3 and CL4 laboratories), we have developed an appropriate inactivation methodology after staining steps, which although results in a reduction of median fluorescent intensity, produces statistically comparable outcomes when judged against non-inactivated samples. This methodology thus extends the use of luminex technology for material that contains highly pathogenic biological agents.
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Phase retrieval by coherent modulation imaging
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhang, Fucai; Chen, Bo; Morrison, Graeme R.
Phase retrieval is a long-standing problem in imaging when only the intensity of the wavefield can be recorded. Coherent diffraction imaging (CDI) is a lensless technique that uses iterative algorithms to recover amplitude and phase contrast images from diffraction intensity data. For general samples, phase retrieval from a single diffraction pattern has been an algorithmic and experimental challenge. Here we report a method of phase retrieval that uses a known modulation of the sample exit-wave. This coherent modulation imaging (CMI) method removes inherent ambiguities of CDI and uses a reliable, rapidly converging iterative algorithm involving three planes. It works formore » extended samples, does not require tight support for convergence, and relaxes dynamic range requirements on the detector. CMI provides a robust method for imaging in materials and biological science, while its single-shot capability will benefit the investigation of dynamical processes with pulsed sources, such as X-ray free electron laser.« less
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Phase retrieval by coherent modulation imaging
Zhang, Fucai; Chen, Bo; Morrison, Graeme R.; ...
2016-11-18
Phase retrieval is a long-standing problem in imaging when only the intensity of the wavefield can be recorded. Coherent diffraction imaging (CDI) is a lensless technique that uses iterative algorithms to recover amplitude and phase contrast images from diffraction intensity data. For general samples, phase retrieval from a single diffraction pattern has been an algorithmic and experimental challenge. Here we report a method of phase retrieval that uses a known modulation of the sample exit-wave. This coherent modulation imaging (CMI) method removes inherent ambiguities of CDI and uses a reliable, rapidly converging iterative algorithm involving three planes. It works formore » extended samples, does not require tight support for convergence, and relaxes dynamic range requirements on the detector. CMI provides a robust method for imaging in materials and biological science, while its single-shot capability will benefit the investigation of dynamical processes with pulsed sources, such as X-ray free electron laser.« less
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Phase retrieval by coherent modulation imaging.
Zhang, Fucai; Chen, Bo; Morrison, Graeme R; Vila-Comamala, Joan; Guizar-Sicairos, Manuel; Robinson, Ian K
2016-11-18
Phase retrieval is a long-standing problem in imaging when only the intensity of the wavefield can be recorded. Coherent diffraction imaging is a lensless technique that uses iterative algorithms to recover amplitude and phase contrast images from diffraction intensity data. For general samples, phase retrieval from a single-diffraction pattern has been an algorithmic and experimental challenge. Here we report a method of phase retrieval that uses a known modulation of the sample exit wave. This coherent modulation imaging method removes inherent ambiguities of coherent diffraction imaging and uses a reliable, rapidly converging iterative algorithm involving three planes. It works for extended samples, does not require tight support for convergence and relaxes dynamic range requirements on the detector. Coherent modulation imaging provides a robust method for imaging in materials and biological science, while its single-shot capability will benefit the investigation of dynamical processes with pulsed sources, such as X-ray free-electron lasers.
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NASA Astrophysics Data System (ADS)
Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun
2016-03-01
Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.
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Plessy, Charles; Desbois, Linda; Fujii, Teruo; Carninci, Piero
2013-02-01
Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells. Copyright © 2013 WILEY Periodicals, Inc.
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Lensfree diffractive tomography for the imaging of 3D cell cultures
NASA Astrophysics Data System (ADS)
Berdeu, Anthony; Momey, Fabien; Dinten, Jean-Marc; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric
2017-02-01
New microscopes are needed to help reaching the full potential of 3D organoid culture studies by gathering large quantitative and systematic data over extended periods of time while preserving the integrity of the living sample. In order to reconstruct large volumes while preserving the ability to catch every single cell, we propose new imaging platforms based on lens-free microscopy, a technic which is addressing these needs in the context of 2D cell culture, providing label-free and non-phototoxic acquisition of large datasets. We built lens-free diffractive tomography setups performing multi-angle acquisitions of 3D organoid cultures embedded in Matrigel and developed dedicated 3D holographic reconstruction algorithms based on the Fourier diffraction theorem. Nonetheless, holographic setups do not record the phase of the incident wave front and the biological samples in Petri dish strongly limit the angular coverage. These limitations introduce numerous artefacts in the sample reconstruction. We developed several methods to overcome them, such as multi-wavelength imaging or iterative phase retrieval. The most promising technic currently developed is based on a regularised inverse problem approach directly applied on the 3D volume to reconstruct. 3D reconstructions were performed on several complex samples such as 3D networks or spheroids embedded in capsules with large reconstructed volumes up to 25 mm3 while still being able to identify single cells. To our knowledge, this is the first time that such an inverse problem approach is implemented in the context of lens-free diffractive tomography enabling to reconstruct large fully 3D volumes of unstained biological samples.
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Bell, Richard W.; Davis, Jerri V.; Femmer, Suzanne R.; Joseph, Robert L.
1997-01-01
Organic-compound samples, including pesticides and semi-volatiles, were collected from 1992-95 at 43 surface-water and 27 bed-sediment and biological-tissue sampling sites within the Ozark Plateaus National Water-Quality Assessment Program study unit. Most surface-water, bed-sediment, and biological-tissue sites have drainage basins predominantly in the Springfield and Salem Plateaus. At most surface-water sampling sites, one to three pesticide samples were collected in the spring and early summer of 1994 and 1995; two sites had additional samples collected either weekly, biweekly, or monthly from February 1994 through December 1994. At most bed-sediment and biological-tissue sampling sites, a single organic-compounds sample was collected. Agricultural pesticide use was approximately 4.9 million pounds of active ingredients per year from 1987-91 in the study unit and was generally greatest in the Springfield and Salem Plateaus pasturelands and in the Osage Plains and Mississippi Alluvial Plain cropland areas. The most frequently applied pesticide in the study unit was 2,4-D. Atrazine was the second most frequently applied pesticide. Corn, pasture, rice, sorghum, and soybeans received approximately 85 percent of the pesticides applied within the study unit. The highest pesticide application rate occurred on these crops in the Mississippi Alluvial and Osage Plains. Pastureland was the crop type that received the greatest amount of pesticides in 53 of the 96 counties in the study unit. The most commonly detected herbicide (63 samples) in surface water was atrazine. Five other pesticides--desethylatrazine, tebuthiuron, prometon, metolachlor, and simazine--were detected in 15 or more samples. The most commonly detected insecticide (13 samples) was p,p'-DDE. Two other insecticides, diazinon and cis-permethrin, were detected in seven or more samples. Pesticides were detected at 39 surface-water sites; samples collected at Yocum Creek near Oak Grove, Ark. had the most pesticide detections (13). Seventeen other sites had samples with six or more pesticide detections. Analysis of pesticide data collected at surface-water sites indicates that the largest variety of different pesticides detected (18) was in small, agricultural drainage basins; the largest percentage of detections of a single pesticide (about 80) was in medium, agricultural basins. Pesticide concentrations were small, and in most cases, at or near the detection limit. Maximum concentrations ranged from 0.001 to 0.007 micrograms per liter (mg/L) at small, forest sites; 0.001 to 0.029 mg/L at medium, forest sites; 0.001 to 0.079 mg/L at small, agricultural sites; and 0.003 to 0.29 mg/L at medium, agricultural sites. Pesticides were detected significantly more often in medium, agricultural basins in the Springfield Plateau. The most commonly detected (13 samples) organic compound in bed sediment, in concentrations noticeably above background levels, was 2,6-dimethylnaphthalene; the maximum concentration of 2,6-dimethylnaphthalene was 130 micrograms per kilogram. Seventeen or more compounds were detected in bed-sediment samples collected at three sites. Four compounds were detected in biological-tissue samples: p,p'-DDT in Corbicula fluminea (Asiatic clam) tissue collected at the Osage River near St. Thomas, Mo. and cis-chlordane, trans-chlordane, and trans-nonachlor in C. fluminea tissue collected at the James River near Boaz, Mo. Organic compounds collected at surface-water, bed-sediment, or biological-tissue sampling sites were not detected in concentrations that exceeded any health criteria or standards. Based on this information, organic compounds do not pose any widespread or persistent problems in the study unit.
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Wang, Yijia; Zeinhom, Mohamed M A; Yang, Mingming; Sun, Rongrong; Wang, Shengfu; Smith, Jordan N; Timchalk, Charles; Li, Lei; Lin, Yuehe; Du, Dan
2017-09-05
Onsite rapid detection of herbicides and herbicide residuals in environmental and biological specimens are important for agriculture, environmental concerns, food safety, and health care. The traditional method for herbicide detection requires expensive laboratory equipment and a long turnaround time. In this work, we developed a single-stripe microliter plate smartphone-based colorimetric device for rapid and low-cost in-field tests. This portable smartphone platform is capable of screening eight samples in a single-stripe microplate. The device combined the advantages of small size (50 × 100 × 160 mm 3 ) and low cost ($10). The platform was calibrated by using two different dye solutions, i.e. methyl blue (MB) and rhodamine B, for the red and green channels. The results showed good correlation with results attained from a traditional laboratory reader. We demonstrated the application of this platform for detection of the herbicide 2,4-dichlorophenoxyacetic acid in the range of 1 to 80 ppb. Spiked samples of tap water, rat serum, plasma, and human serum were tested by our device. Recoveries obtained varied from 95.6% to 105.2% for all of the spiked samples using the microplate reader and from 93.7% to 106.9% for all of the samples using the smartphone device. This work validated that the smartphone optical-sensing platform is comparable to the commercial microplate reader; it is eligible for onsite, rapid, and low-cost detection of herbicides for environmental evaluation and biological monitoring.
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Photon-Counting H33D Detector for Biological Fluorescence Imaging
Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.
2010-01-01
We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:20151021
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Fiducial marker for correlating images
Miller, Lisa Marie [Rocky Point, NY; Smith, Randy J [Wading River, NY; Warren, John B [Port Jefferson, NY; Elliott, Donald [Hampton Bays, NY
2011-06-21
The invention relates to a fiducial marker having a marking grid that is used to correlate and view images produced by different imaging modalities or different imaging and viewing modalities. More specifically, the invention relates to the fiducial marking grid that has a grid pattern for producing either a viewing image and/or a first analytical image that can be overlaid with at least one other second analytical image in order to view a light path or to image different imaging modalities. Depending on the analysis, the grid pattern has a single layer of a certain thickness or at least two layers of certain thicknesses. In either case, the grid pattern is imageable by each imaging or viewing modality used in the analysis. Further, when viewing a light path, the light path of the analytical modality cannot be visualized by viewing modality (e.g., a light microscope objective). By correlating these images, the ability to analyze a thin sample that is, for example, biological in nature but yet contains trace metal ions is enhanced. Specifically, it is desired to analyze both the organic matter of the biological sample and the trace metal ions contained within the biological sample without adding or using extrinsic labels or stains.
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Low cost light-sheet microscopy for whole brain imaging
NASA Astrophysics Data System (ADS)
Kumar, Manish; Nasenbeny, Jordan; Kozorovitskiy, Yevgenia
2018-02-01
Light-sheet microscopy has evolved as an indispensable tool in imaging biological samples. It can image 3D samples at fast speed, with high-resolution optical sectioning, and with reduced photobleaching effects. These properties make light-sheet microscopy ideal for imaging fluorophores in a variety of biological samples and organisms, e.g. zebrafish, drosophila, cleared mouse brains, etc. While most commercial turnkey light-sheet systems are expensive, the existing lower cost implementations, e.g. OpenSPIM, are focused on achieving high-resolution imaging of small samples or organisms like zebrafish. In this work, we substantially reduce the cost of light-sheet microscope system while targeting to image much larger samples, i.e. cleared mouse brains, at single-cell resolution. The expensive components of a lightsheet system - excitation laser, water-immersion objectives, and translation stage - are replaced with an incoherent laser diode, dry objectives, and a custom-built Arduino-controlled translation stage. A low-cost CUBIC protocol is used to clear fixed mouse brain samples. The open-source platforms of μManager and Fiji support image acquisition, processing, and visualization. Our system can easily be extended to multi-color light-sheet microscopy.
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Diffraction Techniques in Structural Biology
Egli, Martin
2016-01-01
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy, and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches, and high-speed computing and visualization, now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail. The present unit combines a general overview of diffraction methods with a detailed description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis, and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide. PMID:27248784
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Diffraction Techniques in Structural Biology
Egli, Martin
2010-01-01
A detailed understanding of chemical and biological function and the mechanisms underlying the activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy and neutron diffraction are well established and have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches and high-speed computing and visualization, now provide specialists and non-specialists alike with a steady flow of molecular images of unprecedented detail. The present chapter combines a general overview of diffraction methods with a step-by-step description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide. PMID:20517991
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Diffraction Techniques in Structural Biology.
Egli, Martin
2016-06-01
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy, and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last twenty years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches, and high-speed computing and visualization, now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail. The present unit combines a general overview of diffraction methods with a detailed description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis, and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.
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Structural Studies of Silver Nanoparticles Obtained Through Single-Step Green Synthesis
NASA Astrophysics Data System (ADS)
Prasad Peddi, Siva; Abdallah Sadeh, Bilal
2015-10-01
Green synthesis of silver Nanoparticles (AGNP's) has been the most prominent among the metallic nanoparticles for research for over a decade and half now due to both the simplicity of preparation and the applicability of biological species with extensive applications in medicine and biotechnology to reduce and trap the particles. The current article uses Eclipta Prostrata leaf extract as the biological species to cap the AGNP's through a single step process. The characterization data obtained was used for the analysis of the sample structure. The article emphasizes the disquisition of their shape and size of the lattice parameters and proposes a general scheme and a mathematical model for the analysis of their dependence. The data of the synthesized AGNP's has been used to advantage through the introduction of a structural shape factor for the crystalline nanoparticles. The properties of the structure of the AGNP's proposed and evaluated through a theoretical model was undeviating with the experimental consequences. This modus operandi gives scope for the structural studies of ultrafine particles prepared using biological methods.
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Triebl, Alexander; Trötzmüller, Martin; Hartler, Jürgen; Stojakovic, Tatjana; Köfeler, Harald C
2018-01-01
An improved approach for selective and sensitive identification and quantitation of lipid molecular species using reversed phase chromatography coupled to high resolution mass spectrometry was developed. The method is applicable to a wide variety of biological matrices using a simple liquid-liquid extraction procedure. Together, this approach combines three selectivity criteria: Reversed phase chromatography separates lipids according to their acyl chain length and degree of unsaturation and is capable of resolving positional isomers of lysophospholipids, as well as structural isomers of diacyl phospholipids and glycerolipids. Orbitrap mass spectrometry delivers the elemental composition of both positive and negative ions with high mass accuracy. Finally, automatically generated tandem mass spectra provide structural insight into numerous glycerolipids, phospholipids, and sphingolipids within a single run. Method validation resulted in a linearity range of more than four orders of magnitude, good values for accuracy and precision at biologically relevant concentration levels, and limits of quantitation of a few femtomoles on column. Hundreds of lipid molecular species were detected and quantified in three different biological matrices, which cover well the wide variety and complexity of various model organisms in lipidomic research. Together with a reliable software package, this method is a prime choice for global lipidomic analysis of even the most complex biological samples. PMID:28415015
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Triebl, Alexander; Trötzmüller, Martin; Hartler, Jürgen; Stojakovic, Tatjana; Köfeler, Harald C
2017-05-15
An improved approach for selective and sensitive identification and quantitation of lipid molecular species using reversed phase chromatography coupled to high resolution mass spectrometry was developed. The method is applicable to a wide variety of biological matrices using a simple liquid-liquid extraction procedure. Together, this approach combines multiple selectivity criteria: Reversed phase chromatography separates lipids according to their acyl chain length and degree of unsaturation and is capable of resolving positional isomers of lysophospholipids, as well as structural isomers of diacyl phospholipids and glycerolipids. Orbitrap mass spectrometry delivers the elemental composition of both positive and negative ions with high mass accuracy. Finally, automatically generated tandem mass spectra provide structural insight into numerous glycerolipids, phospholipids, and sphingolipids within a single run. Calibration showed linearity ranges of more than four orders of magnitude, good values for accuracy and precision at biologically relevant concentration levels, and limits of quantitation of a few femtomoles on column. Hundreds of lipid molecular species were detected and quantified in three different biological matrices, which cover well the wide variety and complexity of various model organisms in lipidomic research. Together with a software package, this method is a prime choice for global lipidomic analysis of even the most complex biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.
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The relevance of time series in molecular ecology and conservation biology.
Habel, Jan C; Husemann, Martin; Finger, Aline; Danley, Patrick D; Zachos, Frank E
2014-05-01
The genetic structure of a species is shaped by the interaction of contemporary and historical factors. Analyses of individuals from the same population sampled at different points in time can help to disentangle the effects of current and historical forces and facilitate the understanding of the forces driving the differentiation of populations. The use of such time series allows for the exploration of changes at the population and intraspecific levels over time. Material from museum collections plays a key role in understanding and evaluating observed population structures, especially if large numbers of individuals have been sampled from the same locations at multiple time points. In these cases, changes in population structure can be assessed empirically. The development of new molecular markers relying on short DNA fragments (such as microsatellites or single nucleotide polymorphisms) allows for the analysis of long-preserved and partially degraded samples. Recently developed techniques to construct genome libraries with a reduced complexity and next generation sequencing and their associated analysis pipelines have the potential to facilitate marker development and genotyping in non-model species. In this review, we discuss the problems with sampling and available marker systems for historical specimens and demonstrate that temporal comparative studies are crucial for the estimation of important population genetic parameters and to measure empirically the effects of recent habitat alteration. While many of these analyses can be performed with samples taken at a single point in time, the measurements are more robust if multiple points in time are studied. Furthermore, examining the effects of habitat alteration, population declines, and population bottlenecks is only possible if samples before and after the respective events are included. © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society.
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Integrated crystal mounting and alignment system for high-throughput biological crystallography
Nordmeyer, Robert A.; Snell, Gyorgy P.; Cornell, Earl W.; Kolbe, William F.; Yegian, Derek T.; Earnest, Thomas N.; Jaklevich, Joseph M.; Cork, Carl W.; Santarsiero, Bernard D.; Stevens, Raymond C.
2007-09-25
A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.
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Integrated crystal mounting and alignment system for high-throughput biological crystallography
Nordmeyer, Robert A.; Snell, Gyorgy P.; Cornell, Earl W.; Kolbe, William; Yegian, Derek; Earnest, Thomas N.; Jaklevic, Joseph M.; Cork, Carl W.; Santarsiero, Bernard D.; Stevens, Raymond C.
2005-07-19
A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.
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Recent advances in magnesium assessment: From single selective sensors to multisensory approach.
Lvova, Larisa; Gonçalves, Carla Guanais; Di Natale, Corrado; Legin, Andrey; Kirsanov, Dmitry; Paolesse, Roberto
2018-03-01
The development of efficient analytical procedures for the selective detection of magnesium is an important analytical task, since this element is one of the most abundant metals in cells and plays an essential role in a plenty of cellular processes. Magnesium misbalance has been related to several pathologies and diseases both in plants and animals, as far as in humans, but the number of suitable methods for magnesium detection especially in life sample and biological environments is scarce. Chemical sensors, due to their high reliability, simplicity of handling and instrumentation, fast and real-time in situ and on site analysis are promising candidates for magnesium analysis and represent an attractive alternative to the standard instrumental methods. Here the recent achievements in the development of chemical sensors for magnesium ions detection over the last decade are reviewed. The working principles and the main types of sensors applied are described. Focus is placed on the optical sensors and multisensory systems applications for magnesium assessment in different media. Further, a critical outlook on the employment of multisensory approach in comparison to single selective sensors application in biological samples is presented. Copyright © 2017 Elsevier B.V. All rights reserved.
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Parallel processing of genomics data
NASA Astrophysics Data System (ADS)
Agapito, Giuseppe; Guzzi, Pietro Hiram; Cannataro, Mario
2016-10-01
The availability of high-throughput experimental platforms for the analysis of biological samples, such as mass spectrometry, microarrays and Next Generation Sequencing, have made possible to analyze a whole genome in a single experiment. Such platforms produce an enormous volume of data per single experiment, thus the analysis of this enormous flow of data poses several challenges in term of data storage, preprocessing, and analysis. To face those issues, efficient, possibly parallel, bioinformatics software needs to be used to preprocess and analyze data, for instance to highlight genetic variation associated with complex diseases. In this paper we present a parallel algorithm for the parallel preprocessing and statistical analysis of genomics data, able to face high dimension of data and resulting in good response time. The proposed system is able to find statistically significant biological markers able to discriminate classes of patients that respond to drugs in different ways. Experiments performed on real and synthetic genomic datasets show good speed-up and scalability.
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Accurate high-speed liquid handling of very small biological samples.
Schober, A; Günther, R; Schwienhorst, A; Döring, M; Lindemann, B F
1993-08-01
Molecular biology techniques require the accurate pipetting of buffers and solutions with volumes in the microliter range. Traditionally, hand-held pipetting devices are used to fulfill these requirements, but many laboratories have also introduced robotic workstations for the handling of liquids. Piston-operated pumps are commonly used in manually as well as automatically operated pipettors. These devices cannot meet the demands for extremely accurate pipetting of very small volumes at the high speed that would be necessary for certain applications (e.g., in sequencing projects with high throughput). In this paper we describe a technique for the accurate microdispensation of biochemically relevant solutions and suspensions with the aid of a piezoelectric transducer. It is suitable for liquids of a viscosity between 0.5 and 500 milliPascals. The obtainable drop sizes range from 5 picoliters to a few nanoliters with up to 10,000 drops per second. Liquids can be dispensed in single or accumulated drops to handle a wide volume range. The system proved to be excellently suitable for the handling of biological samples. It did not show any detectable negative impact on the biological function of dissolved or suspended molecules or particles.
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National Aeronautics and Space Administration Biological Specimen Repository
NASA Technical Reports Server (NTRS)
McMonigal, Kathleen A.; Pietrzyk, Robert a.; Johnson, Mary Anne
2008-01-01
The National Aeronautics and Space Administration Biological Specimen Repository (Repository) is a storage bank that is used to maintain biological specimens over extended periods of time and under well-controlled conditions. Samples from the International Space Station (ISS), including blood and urine, will be collected, processed and archived during the preflight, inflight and postflight phases of ISS missions. This investigation has been developed to archive biosamples for use as a resource for future space flight related research. The International Space Station (ISS) provides a platform to investigate the effects of microgravity on human physiology prior to lunar and exploration class missions. The storage of crewmember samples from many different ISS flights in a single repository will be a valuable resource with which researchers can study space flight related changes and investigate physiological markers. The development of the National Aeronautics and Space Administration Biological Specimen Repository will allow for the collection, processing, storage, maintenance, and ethical distribution of biosamples to meet goals of scientific and programmatic relevance to the space program. Archiving of the biosamples will provide future research opportunities including investigating patterns of physiological changes, analysis of components unknown at this time or analyses performed by new methodologies.
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Biomedical imaging with THz waves
NASA Astrophysics Data System (ADS)
Nguyen, Andrew
2010-03-01
We discuss biomedical imaging using radio waves operating in the terahertz (THz) range between 300 GHz to 3 THz. Particularly, we present the concept for two THz imaging systems. One system employs single antenna, transmitter and receiver operating over multi-THz-frequency simultaneously for sensing and imaging small areas of the human body or biological samples. Another system consists of multiple antennas, a transmitter, and multiple receivers operating over multi-THz-frequency capable of sensing and imaging simultaneously the whole body or large biological samples. Using THz waves for biomedical imaging promises unique and substantial medical benefits including extremely small medical devices, extraordinarily fine spatial resolution, and excellent contrast between images of diseased and healthy tissues. THz imaging is extremely attractive for detection of cancer in the early stages, sensing and imaging of tissues near the skin, and study of disease and its growth versus time.
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NASA Astrophysics Data System (ADS)
Möhler, Christian; Russ, Tom; Wohlfahrt, Patrick; Elter, Alina; Runz, Armin; Richter, Christian; Greilich, Steffen
2018-01-01
An experimental setup for consecutive measurement of ion and x-ray absorption in tissue or other materials is introduced. With this setup using a 3D-printed sample container, the reference stopping-power ratio (SPR) of materials can be measured with an uncertainty of below 0.1%. A total of 65 porcine and bovine tissue samples were prepared for measurement, comprising five samples each of 13 tissue types representing about 80% of the total body mass (three different muscle and fatty tissues, liver, kidney, brain, heart, blood, lung and bone). Using a standard stoichiometric calibration for single-energy CT (SECT) as well as a state-of-the-art dual-energy CT (DECT) approach, SPR was predicted for all tissues and then compared to the measured reference. With the SECT approach, the SPRs of all tissues were predicted with a mean error of (-0.84 ± 0.12)% and a mean absolute error of (1.27 ± 0.12)%. In contrast, the DECT-based SPR predictions were overall consistent with the measured reference with a mean error of (-0.02 ± 0.15)% and a mean absolute error of (0.10 ± 0.15)%. Thus, in this study, the potential of DECT to decrease range uncertainty could be confirmed in biological tissue.
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Dual Raman-Brillouin spectroscopic investigation of plant stress response and development
NASA Astrophysics Data System (ADS)
Coker, Zachary; Troyanova-Wood, Maria; Marble, Kassie; Yakovlev, Vladislav
2018-03-01
Raman and Brillouin spectroscopy are powerful tools for non-invasive and non-destructive investigations of material chemical and mechanical properties. In this study, we use a newly developed custom-built dual Raman-Brillouin microspectroscopy instrument to build on previous works studying in-vivo stress response of live plants using only a Raman spectroscopy system. This dual Raman-Brillouin spectroscopy system is capable of fast simultaneous spectra acquisition from single-point locations. Shifts and changes in a samples Brillouin spectrum indicate a change in the physical characteristics of the sample, namely mechano-elasticity; in measuring this change, we can establish a relationship between the mechanical properties of a sample and known stress response agents, such as reactive oxygen species and other chemical constituents as indicated by peaks in the Raman spectra of the same acquisition point. Simultaneous application of these spectroscopic techniques offers great promise for future development and applications in agricultural and biological studies and can help to improve our understanding of mechanochemical changes of plants and other biological samples in response to environmental and chemically induced stresses at microscopic or cellular level.
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Girshovitz, Pinhas; Frenklach, Irena; Shaked, Natan T
2015-11-01
We propose a new portable imaging configuration that can double the field of view (FOV) of existing off-axis interferometric imaging setups, including broadband off-axis interferometers. This configuration is attached at the output port of the off-axis interferometer and optically creates a multiplexed interferogram on the digital camera, which is composed of two off-axis interferograms with straight fringes at orthogonal directions. Each of these interferograms contains a different FOV of the imaged sample. Due to the separation of these two FOVs in the spatial-frequency domain, they can be fully reconstructed separately, while obtaining two complex wavefronts from the sample at once. Since the optically multiplexed off-axis interferogram is recorded by the camera in a single exposure, fast dynamics can be recorded with a doubled imaging area. We used this technique for quantitative phase microscopy of biological samples with extended FOV. We demonstrate attaching the proposed module to a diffractive phase microscopy interferometer, illuminated by a broadband light source. The biological samples used for the experimental demonstrations include microscopic diatom shells, cancer cells, and flowing blood cells.
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NASA Astrophysics Data System (ADS)
Benktander, John D.; Gizaw, Solomon T.; Gaunitz, Stefan; Novotny, Milos V.
2018-05-01
Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-μg protein starting material or possibly less. These methodological improvements can help accelerating future glycomic comprehensive studies, especially for precious clinical samples.
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Identification of Phosphorylated Proteins on a Global Scale.
Iliuk, Anton
2018-05-31
Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.
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Yu, Feiqiao Brian; Blainey, Paul C; Schulz, Frederik; Woyke, Tanja; Horowitz, Mark A; Quake, Stephen R
2017-01-01
Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell. DOI: http://dx.doi.org/10.7554/eLife.26580.001 PMID:28678007
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Beaulieu, Karen M.; Bell, Amanda H.; Coles, James F.
2012-01-01
Beginning in 1999, the U.S. Geological Survey National Water Quality Assessment Program investigated the effects of urban development on stream ecosystems in nine metropolitan study areas across the United States. In seven of these study areas, stream-chemistry samples were collected every other month for 1 year at 6 to 10 sites. Within a study area, the sites collectively represented a gradient of urban development from minimally to highly developed watersheds, based on the percentage of urban land cover; depending on study area, the land cover before urban development was either forested or agricultural. The stream-chemistry factors measured in the samples were total nitrogen, total phosphorus, chloride, and pesticide toxicity. These data were used to characterize the stream-chemistry factors in four ways (hereafter referred to as characterizations)—seasonal high-flow value, seasonal low-flow value, the median value (representing a single integrated value of the factor over the year), and the standard deviation of values (representing the variation of the factor over the year). Aquatic macroinvertebrate communities were sampled at each site to infer the biological condition of the stream based on the relative sensitivity of the community to environmental stressors. A Spearman correlation analysis was used to evaluate relations between (1) urban development and each characterization of the stream-chemistry factors and (2) the biological condition of a stream and the different characterizations of chloride and pesticide toxicity. Overall, the study areas where the land cover before urban development was primarily forested had a greater number of moderate and strong relations compared with the study areas where the land cover before urban development was primarily agriculture; this was true when urban development was correlated with the stream-chemistry factors (except chloride) and when chloride and pesticide toxicity was correlated with the biological condition. Except for primarily phosphorus in two study areas, stream-chemistry factors generally increased with urban development, and among the different characterizations, the median value typically indicated the strongest relations. The variation in stream-chemistry factors throughout the year generally increased with urban development, indicating that water quality became less consistent as watersheds were developed. In study areas with high annual snow fall, the variation in chloride concentrations throughout the year was particularly strongly related to urban development, likely a result of road salt applications during the winter. The relations of the biological condition to chloride and pesticide toxicity were calculated irrespective of urban development, but the overall results indicated that the relations were still stronger in the study areas that had been forested before urban development. The weaker relations in the study areas that had been agricultural before urban development were likely the results of biological communities having been degraded from agricultural practices in the watersheds. Collectively, these results indicated that, compared with sampling a stream at a single point in time, sampling at regular intervals during a year may provide a more representative measure of water quality, especially in the areas of high urban development where water quality fluctuated more widely between samples. Furthermore, the use of "integrated" values of stream chemistry factors may be more appropriate when assessing relations to the biological condition of a stream because the taxa composition of a biological community typically reflects the water-quality conditions over time.
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Microfluidic single-cell whole-transcriptome sequencing.
Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi
2014-05-13
Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.
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Fast method of cross-talk effect reduction in biomedical imaging (Conference Presentation)
NASA Astrophysics Data System (ADS)
Nowakowski, Maciej; Kolenderska, Sylwia M.; Borycki, Dawid; Wojtkowski, Maciej
2016-03-01
Optical imaging of biological samples or living tissue structures requires light delivery to a region of interest and then collection of scattered light or fluorescent light in order to reconstruct an image of the object. When the coherent illumination light enters bulky biological object, each of scattering center (single molecule, group of molecules or other sample feature) acts as a secondary light source. As a result, scattered spherical waves from these secondary sources interact with each other, generating cross-talk noise between optical channels (eigenmodes). The cross-talk effect have serious impact on the performance of the imaging systems. In particular it reduces an ability of optical system to transfer high spatial frequencies thereby reducing its resolution. In this work we present a fast method to eliminate all unwanted waves combination, that overlap at image plane, suppressing recovery of high spatial frequencies by using the spatio-temporal optical coherence manipulation (STOC, [1]). In this method a number of phase mask is introduced to illuminating beam by spatial light modulator in a time of single image acquisition. We use a digital mirror device (DMD) in order to rapid cross-talk noise reduction (up to 22kHz modulation frequency) when imaging living biological cells in vivo by using full-field microscopy setup with double pass arrangement. This, to our best knowledge, has never been shown before. [1] D. Borycki, M. Nowakowski, and M. Wojtkowski, Opt. Lett. 38, 4817 (2013).
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Database Resources of the BIG Data Center in 2018
Xu, Xingjian; Hao, Lili; Zhu, Junwei; Tang, Bixia; Zhou, Qing; Song, Fuhai; Chen, Tingting; Zhang, Sisi; Dong, Lili; Lan, Li; Wang, Yanqing; Sang, Jian; Hao, Lili; Liang, Fang; Cao, Jiabao; Liu, Fang; Liu, Lin; Wang, Fan; Ma, Yingke; Xu, Xingjian; Zhang, Lijuan; Chen, Meili; Tian, Dongmei; Li, Cuiping; Dong, Lili; Du, Zhenglin; Yuan, Na; Zeng, Jingyao; Zhang, Zhewen; Wang, Jinyue; Shi, Shuo; Zhang, Yadong; Pan, Mengyu; Tang, Bixia; Zou, Dong; Song, Shuhui; Sang, Jian; Xia, Lin; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Zhang, Yang; Sheng, Xin; Lu, Mingming; Wang, Qi; Xiao, Jingfa; Zou, Dong; Wang, Fan; Hao, Lili; Liang, Fang; Li, Mengwei; Sun, Shixiang; Zou, Dong; Li, Rujiao; Yu, Chunlei; Wang, Guangyu; Sang, Jian; Liu, Lin; Li, Mengwei; Li, Man; Niu, Guangyi; Cao, Jiabao; Sun, Shixiang; Xia, Lin; Yin, Hongyan; Zou, Dong; Xu, Xingjian; Ma, Lina; Chen, Huanxin; Sun, Yubin; Yu, Lei; Zhai, Shuang; Sun, Mingyuan; Zhang, Zhang; Zhao, Wenming; Xiao, Jingfa; Bao, Yiming; Song, Shuhui; Hao, Lili; Li, Rujiao; Ma, Lina; Sang, Jian; Wang, Yanqing; Tang, Bixia; Zou, Dong; Wang, Fan
2018-01-01
Abstract The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. PMID:29036542
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Watanabe, Manabu; Kusano, Junko; Ohtaki, Shinsaku; Ishikura, Takashi; Katayama, Jin; Koguchi, Akira; Paumen, Michael; Hayashi, Yoshiharu
2014-09-01
Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈ 10(6) cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 10(31-35). For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.
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NASA Astrophysics Data System (ADS)
Engel, Andreas; Miles, Mervyn
2008-09-01
The atomic force microscope (AFM) and related scanning probe microscopes have become resourceful tools to study cells, supramolecular assemblies and single biomolecules, because they allow investigations of such structures in native environments. Quantitative information has been gathered about the surface structure of membrane proteins to lateral and vertical resolutions of 0.5 nm and 0.1 nm, respectively, about the forces that keep protein-protein and protein-nucleic acid assemblies together as well as single proteins in their native conformation, and about the nanomechanical properties of cells in health and disease. Such progress has been achieved mainly because of constant development of AFM instrumentation and sample preparation methods. This special issue of Nanotechnology presents papers from leading laboratories in the field of nanobiology, covering a wide range of topics in the form of original and novel scientific contributions. It addresses achievements in instrumentation, sample preparation, automation and in biological applications. These papers document the creativity and persistence of researchers pursuing the goal to unravel the structure and dynamics of cells, supramolecuar structures and single biomolecules at work. Improved cantilever sensors, novel optical probes, and quantitative data on supports for electrochemical experiments open new avenues for characterizing biological nanomachines down to the single molecule. Comparative measurements of healthy and metastatic cells promise new methods for early detection of tumors, and possible assessments of drug efficacy. High-speed AFMs document possibilities to monitor crystal growth and to observe large structures at video rate. A wealth of information on amyloid-type fibers as well as on membrane proteins has been gathered by single molecule force spectroscopy—a technology now being automated for large-scale data collection. With the progress of basic research and a strong industry supporting instrumentation development by improving robustness and reliability and making new instruments available to the community, nanobiology has the potential to develop into a field with great impact on our understanding of the complexity of life, and to provide a major contribution to human health. This special issue of Nanotechnology on nanobiology would not have been possible without the highly professional support from Nina Couzin, Amy Harvey and the Nanotechnology team at IOP Publishing. We are thankful for their most constructive and effective help in pushing the project forward. We are also thankful to all the authors who have contributed with excellent original articles, as well as to the referees who have helped to make this special issue such an insightful document of a rapidly moving field.
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Zhang, Xiaomeng; Shao, Bin; Wu, Yangle; Qi, Ouyang
2013-01-01
One of the major objectives in systems biology is to understand the relation between the topological structures and the dynamics of biological regulatory networks. In this context, various mathematical tools have been developed to deduct structures of regulatory networks from microarray expression data. In general, from a single data set, one cannot deduct the whole network structure; additional expression data are usually needed. Thus how to design a microarray expression experiment in order to get the most information is a practical problem in systems biology. Here we propose three methods, namely, maximum distance method, trajectory entropy method, and sampling method, to derive the optimal initial conditions for experiments. The performance of these methods is tested and evaluated in three well-known regulatory networks (budding yeast cell cycle, fission yeast cell cycle, and E. coli. SOS network). Based on the evaluation, we propose an efficient strategy for the design of microarray expression experiments.
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Ensemble of sparse classifiers for high-dimensional biological data.
Kim, Sunghan; Scalzo, Fabien; Telesca, Donatello; Hu, Xiao
2015-01-01
Biological data are often high in dimension while the number of samples is small. In such cases, the performance of classification can be improved by reducing the dimension of data, which is referred to as feature selection. Recently, a novel feature selection method has been proposed utilising the sparsity of high-dimensional biological data where a small subset of features accounts for most variance of the dataset. In this study we propose a new classification method for high-dimensional biological data, which performs both feature selection and classification within a single framework. Our proposed method utilises a sparse linear solution technique and the bootstrap aggregating algorithm. We tested its performance on four public mass spectrometry cancer datasets along with two other conventional classification techniques such as Support Vector Machines and Adaptive Boosting. The results demonstrate that our proposed method performs more accurate classification across various cancer datasets than those conventional classification techniques.
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NASA Biological Specimen Repository
NASA Technical Reports Server (NTRS)
Pietrzyk, Robert; McMonigal, K. A.; Sams, C. F.; Johnson, M. A.
2009-01-01
The NASA Biological Specimen Repository (NBSR) has been established to collect, process, annotate, store, and distribute specimens under the authority of the NASA/JSC Committee for the Protection of Human Subjects. The International Space Station (ISS) provides a platform to investigate the effects of microgravity on human physiology prior to lunar and exploration class missions. The NBSR is a secure controlled storage facility that is used to maintain biological specimens over extended periods of time, under well-controlled conditions, for future use in approved human spaceflight-related research protocols. The repository supports the Human Research Program, which is charged with identifying and investigating physiological changes that occur during human spaceflight, and developing and implementing effective countermeasures when necessary. The storage of crewmember samples from many different ISS flights in a single repository will be a valuable resource with which researchers can validate clinical hypotheses, study space-flight related changes, and investigate physiological markers All samples collected require written informed consent from each long duration crewmember. The NBSR collects blood and urine samples from all participating long duration ISS crewmembers. These biological samples are collected pre-flight at approximately 45 days prior to launch, during flight on flight days 15, 30, 60 120 and within 2 weeks of landing. Postflight sessions are conducted 3 and 30 days following landing. The number of inflight sessions is dependent on the duration of the mission. Operations began in 2007 and as of October 2009, 23 USOS crewmembers have completed or agreed to participate in this project. As currently planned, these human biological samples will be collected from crewmembers covering multiple ISS missions until the end of U.S. presence on the ISS or 2017. The NBSR will establish guidelines for sample distribution that are consistent with ethical principles, protection of crewmember confidentiality, prevailing laws and regulations, intellectual property policies, and consent form language. A NBSR Advisory Board composed of representatives of all participating agencies will be established to evaluate each request by an investigator for use of the samples to ensure the request reflects the mission of the NBSR.
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Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta
2017-10-01
The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.
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Fast, reagentless and reliable screening of "white powders" during the bioterrorism hoaxes.
Włodarski, Maksymilian; Kaliszewski, Miron; Trafny, Elżbieta Anna; Szpakowska, Małgorzata; Lewandowski, Rafał; Bombalska, Aneta; Kwaśny, Mirosław; Kopczyński, Krzysztof; Mularczyk-Oliwa, Monika
2015-03-01
The classification of dry powder samples is an important step in managing the consequences of terrorist incidents. Fluorescence decays of these samples (vegetative bacteria, bacterial endospores, fungi, albumins and several flours) were measured with stroboscopic technique using an EasyLife LS system PTI. Three pulsed nanosecond LED sources, generating 280, 340 and 460nm were employed for samples excitation. The usefulness of a new 460nm light source for fluorescence measurements of dry microbial cells has been demonstrated. The principal component analysis (PCA) and hierarchical cluster analysis (HCA) have been used for classification of dry biological samples. It showed that the single excitation wavelength was not sufficient for differentiation of biological samples of diverse origin. However, merging fluorescence decays from two or three excitation wavelengths allowed classification of these samples. An experimental setup allowing the practical implementation of this method for the real time fluorescence decay measurement was designed. It consisted of the LED emitting nanosecond pulses at 280nm and two fast photomultiplier tubes (PMTs) for signal detection in two fluorescence bands simultaneously. The positive results of the dry powder samples measurements confirmed that the fluorescence decay-based technique could be a useful tool for fast classification of the suspected "white powders" performed by the first responders. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Electrostatic sampling of trace DNA from clothing.
Zieger, Martin; Defaux, Priscille Merciani; Utz, Silvia
2016-05-01
During acts of physical aggression, offenders frequently come into contact with clothes of the victim, thereby leaving traces of DNA-bearing biological material on the garments. Since tape-lifting and swabbing, the currently established methods for non-destructive trace DNA sampling from clothing, both have their shortcomings in collection efficiency and handling, we thought about a new collection method for these challenging samples. Testing two readily available electrostatic devices for their potential to sample biological material from garments made of different fabrics, we found one of them, the electrostatic dust print lifter (DPL), to perform comparable to well-established sampling with wet cotton swabs. In simulated aggression scenarios, we had the same success rate for the establishment of single aggressor profiles, suitable for database submission, with both the DPL and wet swabbing. However, we lost a substantial amount of information with electrostatic sampling, since almost no mixed aggressor-victim profiles suitable for database entry could be established, compared to conventional swabbing. This study serves as a proof of principle for electrostatic DNA sampling from items of clothing. The technique still requires optimization before it might be used in real casework. But we are confident that in the future it could be an efficient and convenient contribution to the toolbox of forensic practitioners.
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NASA Astrophysics Data System (ADS)
Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto
2017-02-01
Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.
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Lavagnino, Zeno; Sancataldo, Giuseppe; d’Amora, Marta; Follert, Philipp; De Pietri Tonelli, Davide; Diaspro, Alberto; Cella Zanacchi, Francesca
2016-01-01
In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces. PMID:27033347
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Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction
NASA Astrophysics Data System (ADS)
Miao, Jianwei; Hodgson, Keith O.; Ishikawa, Tetsuya; Larabell, Carolyn A.; Legros, Mark A.; Nishino, Yoshinori
2003-01-01
We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 Å. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.
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Li, Ming; Ohi, Kazutaka; Chen, Chunhui; He, Qinghua; Liu, Jie-Wei; Chen, Chuansheng; Luo, Xiong-Jian; Dong, Qi; Hashimoto, Ryota; Su, Bing
2014-12-01
Hippocampal volume is a key brain structure for learning ability and memory process, and hippocampal atrophy is a recognized biological marker of Alzheimer's disease. However, the genetic bases of hippocampal volume are still unclear although it is a heritable trait. Genome-wide association studies (GWASs) on hippocampal volume have implicated several significantly associated genetic variants in Europeans. Here, to test the contributions of these GWASs identified genetic variants to hippocampal volume in different ethnic populations, we screened the GWAS-identified candidate single-nucleotide polymorphisms in 3 independent healthy Asian brain imaging samples (a total of 990 subjects). The results showed that none of these single-nucleotide polymorphisms were associated with hippocampal volume in either individual or combined Asian samples. The replication results suggested a complexity of genetic architecture for hippocampal volume and potential genetic heterogeneity between different ethnic populations. Copyright © 2014 Elsevier Inc. All rights reserved.
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Wang, Zhuo; Jin, Shuilin; Liu, Guiyou; Zhang, Xiurui; Wang, Nan; Wu, Deliang; Hu, Yang; Zhang, Chiping; Jiang, Qinghua; Xu, Li; Wang, Yadong
2017-05-23
The development of single-cell RNA sequencing has enabled profound discoveries in biology, ranging from the dissection of the composition of complex tissues to the identification of novel cell types and dynamics in some specialized cellular environments. However, the large-scale generation of single-cell RNA-seq (scRNA-seq) data collected at multiple time points remains a challenge to effective measurement gene expression patterns in transcriptome analysis. We present an algorithm based on the Dynamic Time Warping score (DTWscore) combined with time-series data, that enables the detection of gene expression changes across scRNA-seq samples and recovery of potential cell types from complex mixtures of multiple cell types. The DTWscore successfully classify cells of different types with the most highly variable genes from time-series scRNA-seq data. The study was confined to methods that are implemented and available within the R framework. Sample datasets and R packages are available at https://github.com/xiaoxiaoxier/DTWscore .
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Absorption spectroscopy at the ultimate quantum limit from single-photon states
NASA Astrophysics Data System (ADS)
Whittaker, R.; Erven, C.; Neville, A.; Berry, M.; O'Brien, J. L.; Cable, H.; Matthews, J. C. F.
2017-02-01
Absorption spectroscopy is routinely used to characterise chemical and biological samples. For the state-of-the-art in laser absorption spectroscopy, precision is theoretically limited by shot-noise due to the fundamental Poisson-distribution of photon number in laser radiation. In practice, the shot-noise limit can only be achieved when all other sources of noise are eliminated. Here, we use wavelength-correlated and tuneable photon pairs to demonstrate how absorption spectroscopy can be performed with precision beyond the shot-noise limit and near the ultimate quantum limit by using the optimal probe for absorption measurement—single photons. We present a practically realisable scheme, which we characterise both the precision and accuracy of by measuring the response of a control feature. We demonstrate that the technique can successfully probe liquid samples and using two spectrally similar types of haemoglobin we show that obtaining a given precision in resolution requires fewer heralded single probe photons compared to using an idealised laser.
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Kogut, Katherine; Eisen, Ellen A.; Jewell, Nicholas P.; Quirós-Alcalá, Lesliam; Castorina, Rosemary; Chevrier, Jonathan; Holland, Nina T.; Barr, Dana Boyd; Kavanagh-Baird, Geri; Eskenazi, Brenda
2012-01-01
Background: Dialkyl phosphate (DAP) metabolites in spot urine samples are frequently used to characterize children’s exposures to organophosphorous (OP) pesticides. However, variable exposure and short biological half-lives of OP pesticides could result in highly variable measurements, leading to exposure misclassification. Objective: We examined within- and between-child variability in DAP metabolites in urine samples collected during 1 week. Methods: We collected spot urine samples over 7 consecutive days from 25 children (3–6 years of age). On two of the days, we collected 24-hr voids. We assessed the reproducibility of urinary DAP metabolite concentrations and evaluated the sensitivity and specificity of spot urine samples as predictors of high (top 20%) or elevated (top 40%) weekly average DAP metabolite concentrations. Results: Within-child variance exceeded between-child variance by a factor of two to eight, depending on metabolite grouping. Although total DAP concentrations in single spot urine samples were moderately to strongly associated with concentrations in same-day 24-hr samples (r ≈ 0.6–0.8, p < 0.01), concentrations in spot samples collected > 1 day apart and in 24-hr samples collected 3 days apart were weakly correlated (r ≈ –0.21 to 0.38). Single spot samples predicted high (top 20%) and elevated (top 40%) full-week average total DAP excretion with only moderate sensitivity (≈ 0.52 and ≈ 0.67, respectively) but relatively high specificity (≈ 0.88 and ≈ 0.78, respectively). Conclusions: The high variability we observed in children’s DAP metabolite concentrations suggests that single-day urine samples provide only a brief snapshot of exposure. Sensitivity analyses suggest that classification of cumulative OP exposure based on spot samples is prone to type 2 classification errors. PMID:23052012
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Vergucht, Eva; Brans, Toon; Beunis, Filip; Garrevoet, Jan; Bauters, Stephen; De Rijcke, Maarten; Deruytter, David; Janssen, Colin; Riekel, Christian; Burghammer, Manfred; Vincze, Laszlo
2015-07-01
Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.
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Nelson, LaRon E.; Morrison-Beedy, Dianne; Kearney, Margaret H.; Dozier, Ann
2013-01-01
Objective The objective of this study was to understand single Black adolescent mothers’ perspectives on the sexual and parenting related aspects of their relationships with the biological fathers of their children. Methods The study was a qualitative description of perspectives from a convenience sample of Black single (non married) adolescent mothers. Data were generated through focus groups and interviews. Participants were recruited using self-referral and health provider referrals. Setting The study was conducted in a county public health department sexually transmitted diseases clinic in Rochester, New York. Participants Single mothers (n=31) ages 15–19 participated in the study. The mean age of participants was 17.5 years (SD 1.4). Findings Four themes were identified that reflected the major characteristics of the relationships between the mothers and the biological fathers of their children: (1) You will always care about your baby daddy because of your child, (2) Negative behavior is tolerated to keep the family together, (3) The baby daddy can get sex as long as we are not on bad terms, and (4) He will always be part of our life. Conclusion Black adolescent mothers have complex relationships with the biological fathers of their children that may include ongoing sexual activity. The intersection of co-parenting and sexual health needs among adolescent mothers highlights the importance of integrating sexually transmitted infections prevention with perinatal health programs. It is important to consider this unique co-parenting relationship when providing risk-reduction counseling to young mothers. PMID:22834723
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Nelson, LaRon E; Morrison-Beedy, Dianne; Kearney, Margaret H; Dozier, Ann
2012-01-01
To understand single Black adolescent mothers' perspectives on the sexual and parenting-related aspects of their relationships with the biological fathers of their children. The study was a qualitative description of perspectives from a convenience sample of Black single (nonmarried) adolescent mothers. Data were generated through focus groups and interviews. Participants were recruited using self-referral and health provider referrals. The study was conducted in a county public health department sexually transmitted diseases clinic in Rochester, New York. Single mothers (N = 31) ages 15 to 19 participated in the study. The mean age of participants was 17.5 years (SD = 1.4). Four themes were identified that reflected the major characteristics of the relationships between the mothers and the biological fathers of their children: (a) You will always care about your "baby daddy" because of your child, (b) Negative behavior is tolerated to keep the family together, (c) The "baby daddy" can get sex as long as we are not on bad terms, and (d) He will always be part of our lives. Black adolescent mothers have complex relationships with the biological fathers of their children that may include ongoing sexual activity. The intersection of coparenting and sexual health needs among adolescent mothers highlights the importance of integrating sexually transmitted infections prevention with perinatal health programs. It is important to consider this unique coparenting relationship when providing risk-reduction counseling to young mothers. © 2012 AWHONN, the Association of Women's Health, Obstetric and Neonatal Nurses.
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Mannion, Philip D; Upchurch, Paul; Carrano, Matthew T; Barrett, Paul M
2011-02-01
The accurate reconstruction of palaeobiodiversity patterns is central to a detailed understanding of the macroevolutionary history of a group of organisms. However, there is increasing evidence that diversity patterns observed directly from the fossil record are strongly influenced by fluctuations in the quality of our sampling of the rock record; thus, any patterns we see may reflect sampling biases, rather than genuine biological signals. Previous dinosaur diversity studies have suggested that fluctuations in sauropodomorph palaeobiodiversity reflect genuine biological signals, in comparison to theropods and ornithischians whose diversity seems to be largely controlled by the rock record. Most previous diversity analyses that have attempted to take into account the effects of sampling biases have used only a single method or proxy: here we use a number of techniques in order to elucidate diversity. A global database of all known sauropodomorph body fossil occurrences (2024) was constructed. A taxic diversity curve for all valid sauropodomorph genera was extracted from this database and compared statistically with several sampling proxies (rock outcrop area and dinosaur-bearing formations and collections), each of which captures a different aspect of fossil record sampling. Phylogenetic diversity estimates, residuals and sample-based rarefaction (including the first attempt to capture 'cryptic' diversity in dinosaurs) were implemented to investigate further the effects of sampling. After 'removal' of biases, sauropodomorph diversity appears to be genuinely high in the Norian, Pliensbachian-Toarcian, Bathonian-Callovian and Kimmeridgian-Tithonian (with a small peak in the Aptian), whereas low diversity levels are recorded for the Oxfordian and Berriasian-Barremian, with the Jurassic/Cretaceous boundary seemingly representing a real diversity trough. Observed diversity in the remaining Triassic-Jurassic stages appears to be largely driven by sampling effort. Late Cretaceous diversity is difficult to elucidate and it is possible that this interval remains relatively under-sampled. Despite its distortion by sampling biases, much of sauropodomorph palaeobiodiversity can be interpreted as a reflection of genuine biological signals, and fluctuations in sea level may account for some of these diversity patterns. © 2010 The Authors. Biological Reviews © 2010 Cambridge Philosophical Society.
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Diagnosing phenotypes of single-sample individuals by edge biomarkers.
Zhang, Wanwei; Zeng, Tao; Liu, Xiaoping; Chen, Luonan
2015-06-01
Network or edge biomarkers are a reliable form to characterize phenotypes or diseases. However, obtaining edges or correlations between molecules for an individual requires measurement of multiple samples of that individual, which are generally unavailable in clinical practice. Thus, it is strongly demanded to diagnose a disease by edge or network biomarkers in one-sample-for-one-individual context. Here, we developed a new computational framework, EdgeBiomarker, to integrate edge and node biomarkers to diagnose phenotype of each single test sample. By applying the method to datasets of lung and breast cancer, it reveals new marker genes/gene-pairs and related sub-networks for distinguishing earlier and advanced cancer stages. Our method shows advantages over traditional methods: (i) edge biomarkers extracted from non-differentially expressed genes achieve better cross-validation accuracy of diagnosis than molecule or node biomarkers from differentially expressed genes, suggesting that certain pathogenic information is only present at the level of network and under-estimated by traditional methods; (ii) edge biomarkers categorize patients into low/high survival rate in a more reliable manner; (iii) edge biomarkers are significantly enriched in relevant biological functions or pathways, implying that the association changes in a network, rather than expression changes in individual molecules, tend to be causally related to cancer development. The new framework of edge biomarkers paves the way for diagnosing diseases and analyzing their molecular mechanisms by edges or networks in one-sample-for-one-individual basis. This also provides a powerful tool for precision medicine or big-data medicine. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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Burnum-Johnson, Kristin E; Kyle, Jennifer E; Eisfeld, Amie J; Casey, Cameron P; Stratton, Kelly G; Gonzalez, Juan F; Habyarimana, Fabien; Negretti, Nicholas M; Sims, Amy C; Chauhan, Sadhana; Thackray, Larissa B; Halfmann, Peter J; Walters, Kevin B; Kim, Young-Mo; Zink, Erika M; Nicora, Carrie D; Weitz, Karl K; Webb-Robertson, Bobbie-Jo M; Nakayasu, Ernesto S; Ahmer, Brian; Konkel, Michael E; Motin, Vladimir; Baric, Ralph S; Diamond, Michael S; Kawaoka, Yoshihiro; Waters, Katrina M; Smith, Richard D; Metz, Thomas O
2017-01-26
The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes.
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Burnum-Johnson, Kristin E.; Kyle, Jennifer E.; Eisfeld, Amie J.; Casey, Cameron P.; Stratton, Kelly G.; Gonzalez, Juan F.; Habyarimana, Fabien; Negretti, Nicholas M.; Sims, Amy C.; Chauhan, Sadhana; Thackray, Larissa B.; Halfmann, Peter J.; Walters, Kevin B.; Kim, Young-Mo; Zink, Erika M.; Nicora, Carrie D.; Weitz, Karl K.; Webb-Robertson, Bobbie-Jo M.; Nakayasu, Ernesto S.; Ahmer, Brian; Konkel, Michael E.; Motin, Vladimir; Baric, Ralph S.; Diamond, Michael S.; Kawaoka, Yoshihiro; Waters, Katrina M.; Smith, Richard D.; Metz, Thomas O.
2017-01-01
The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes. PMID:28091625
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Jung, Seung-Ryoung; Han, Rui; Sun, Wei; Jiang, Yifei; Fujimoto, Bryant S; Yu, Jiangbo; Kuo, Chun-Ting; Rong, Yu; Zhou, Xing-Hua; Chiu, Daniel T
2018-05-15
We describe here a flow platform for quantifying the number of biomolecules on individual fluorescent nanoparticles. The platform combines line-confocal fluorescence detection with near nanoscale channels (1-2 μm in width and height) to achieve high single-molecule detection sensitivity and throughput. The number of biomolecules present on each nanoparticle was determined by deconvolving the fluorescence intensity distribution of single-nanoparticle-biomolecule complexes with the intensity distribution of single biomolecules. We demonstrate this approach by quantifying the number of streptavidins on individual semiconducting polymer dots (Pdots); streptavidin was rendered fluorescent using biotin-Alexa647. This flow platform has high-throughput (hundreds to thousands of nanoparticles detected per second) and requires minute amounts of sample (∼5 μL at a dilute concentration of 10 pM). This measurement method is an additional tool for characterizing synthetic or biological nanoparticles.
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Single-cylinder diesel engine study of four vegetable oils
DOE Office of Scientific and Technical Information (OSTI.GOV)
Jacobus, M.J.; Geyer, S.M.; Lestz, S.S.
A single-cylinder, 0.36l, D.I. Diesel engine was operated on Diesel fuel, sunflowerseed oil, cottonseed oil, soybean oil, and peanut oil. The purpose of this study was to provide a detailed comparison of performance and emissions data and to characterize the biological activity of the particulate soluble organic fraction for each fuel using the Ames Salmonella typhimurium test. In addition, exhaust gas aldehyde samples were collected using the DNPH method. These samples were analyzed gravimetrically and separated into components from formaldehyde to heptaldehyde with a gas chromatograph. Results comparing the vegetable oils to Diesel fuel generally show slight improvements in thermalmore » efficiency and indicated specific energy consumption; equal or higher gas-phase emissions; lower indicated specific revertant emissions; and significantly higher aldehyde emissions, including an increased percentage of formaldehyde.« less
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Sauer, Eva; Reinke, Ann-Kathrin; Courts, Cornelius
2016-05-01
Applying molecular genetic approaches for the identification of forensically relevant body fluids, which often yield crucial information for the reconstruction of a potential crime, is a current topic of forensic research. Due to their body fluid specific expression patterns and stability against degradation, microRNAs (miRNA) emerged as a promising molecular species, with a range of candidate markers published. The analysis of miRNA via quantitative Real-Time PCR, however, should be based on a relevant strategy of normalization of non-biological variances to deliver reliable and biologically meaningful results. The herein presented work is the as yet most comprehensive study of forensic body fluid identification via miRNA expression analysis based on a thoroughly validated qPCR procedure and unbiased statistical decision making to identify single source samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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X-ray propagation microscopy of biological cells using waveguides as a quasipoint source
DOE Office of Scientific and Technical Information (OSTI.GOV)
Giewekemeyer, K.; Krueger, S. P.; Kalbfleisch, S.
2011-02-15
We have used x-ray waveguides as highly confining optical elements for nanoscale imaging of unstained biological cells using the simple geometry of in-line holography. The well-known twin-image problem is effectively circumvented by a simple and fast iterative reconstruction. The algorithm which combines elements of the classical Gerchberg-Saxton scheme and the hybrid-input-output algorithm is optimized for phase-contrast samples, well-justified for imaging of cells at multi-keV photon energies. The experimental scheme allows for a quantitative phase reconstruction from a single holographic image without detailed knowledge of the complex illumination function incident on the sample, as demonstrated for freeze-dried cells of the eukaryoticmore » amoeba Dictyostelium discoideum. The accessible resolution range is explored by simulations, indicating that resolutions on the order of 20 nm are within reach applying illumination times on the order of minutes at present synchrotron sources.« less
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Cvrčková, Fatima; Luštinec, Jiří; Žárský, Viktor
2015-01-01
We usually expect the dose-response curves of biological responses to quantifiable stimuli to be simple, either monotonic or exhibiting a single maximum or minimum. Deviations are often viewed as experimental noise. However, detailed measurements in plant primary tissue cultures (stem pith explants of kale and tobacco) exposed to varying doses of sucrose, cytokinins (BA or kinetin) or auxins (IAA or NAA) revealed that growth and several biochemical parameters exhibit multiple reproducible, statistically significant maxima over a wide range of exogenous substance concentrations. This results in complex, non-monotonic dose-response curves, reminiscent of previous reports of analogous observations in both metazoan and plant systems responding to diverse pharmacological treatments. These findings suggest the existence of a hitherto neglected class of biological phenomena resulting in dose-response curves exhibiting periodic patterns of maxima and minima, whose causes remain so far uncharacterized, partly due to insufficient sampling frequency used in many studies.
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Single Biomolecules at Cryogenic Temperatures: From Structure to Dynamics
NASA Astrophysics Data System (ADS)
Hofmann, Clemens; Kulzer, Florian; Zondervan, Rob; Köhler, Jürgen; Orrit, Michel
Elucidating the dynamics of proteins remains a central and daunting challenge of molecular biology. In our contribution we discuss the relevance of lowtemperature observations not only to structure, but also to dynamics, and thereby to the function of proteins. We first review investigations on light-harvesting complexes to illustrate how increased photostability at low temperatures and spectral selection provide a deeper insight into the excitonic interactions of the chromophores and the dynamics of the protein scaffold. Furthermore, we introduce a novel technique that achieves controlled, reproducible temperature cycles of a microscopic sample on microsecond timescales. We discuss the potential of this technique as a tool to achieve repeatable single-molecule freeze-trapping and to overcome some of the limitations of single-molecule experiments at room temperature.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Boedicker, J.; Li, L; Kline, T
2008-01-01
This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminatingmore » the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as stochastic confinement. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.« less
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Carlisle, Daren M.; Bryant, Wade L.
2011-01-01
Many physicochemical factors potentially impair stream ecosystems in urbanizing basins, but few studies have evaluated their relative importance simultaneously, especially in different environmental settings. We used data collected in 25 to 30 streams along a gradient of urbanization in each of 6 metropolitan areas (MAs) to evaluate the relative importance of 11 physicochemical factors on the condition of algal, macroinvertebrate, and fish assemblages. For each assemblage, biological condition was quantified using 2 separate metrics, nonmetric multidimensional scaling ordination site scores and the ratio of observed/expected taxa, both derived in previous studies. Separate linear regression models with 1 or 2 factors as predictors were developed for each MA and assemblage metric. Model parsimony was evaluated based on Akaike’s Information Criterion for small sample size (AICc) and Akaike weights, and variable importance was estimated by summing the Akaike weights across models containing each stressor variable. Few of the factors were strongly correlated (Pearson |r| > 0.7) within MAs. Physicochemical factors explained 17 to 81% of variance in biological condition. Most (92 of 118) of the most plausible models contained 2 predictors, and generally more variance could be explained by the additive effects of 2 factors than by any single factor alone. None of the factors evaluated was universally important for all MAs or biological assemblages. The relative importance of factors varied for different measures of biological condition, biological assemblages, and MA. Our results suggest that the suite of physicochemical factors affecting urban stream ecosystems varies across broad geographic areas, along gradients of urban intensity, and among basins within single MAs.
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A single-image method for x-ray refractive index CT.
Mittone, A; Gasilov, S; Brun, E; Bravin, A; Coan, P
2015-05-07
X-ray refraction-based computer tomography imaging is a well-established method for nondestructive investigations of various objects. In order to perform the 3D reconstruction of the index of refraction, two or more raw computed tomography phase-contrast images are usually acquired and combined to retrieve the refraction map (i.e. differential phase) signal within the sample. We suggest an approximate method to extract the refraction signal, which uses a single raw phase-contrast image. This method, here applied to analyzer-based phase-contrast imaging, is employed to retrieve the index of refraction map of a biological sample. The achieved accuracy in distinguishing the different tissues is comparable with the non-approximated approach. The suggested procedure can be used for precise refraction computer tomography with the advantage of a reduction of at least a factor of two of both the acquisition time and the dose delivered to the sample with respect to any of the other algorithms in the literature.
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Sun, Dayong; Cree, Melanie G; Zhang, Xiao-Jun; Bøersheim, Elisabet; Wolfe, Robert R
2006-02-01
We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.
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Low-kilovolt coherent electron diffractive imaging instrument based on a single-atom electron source
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lin, Chun-Yueh; Chang, Wei-Tse; Chen, Yi-Sheng
2016-03-15
In this work, a transmission-type, low-kilovolt coherent electron diffractive imaging instrument was constructed. It comprised a single-atom field emitter, a triple-element electrostatic lens, a sample holder, and a retractable delay line detector to record the diffraction patterns at different positions behind the sample. It was designed to image materials thinner than 3 nm. The authors analyzed the asymmetric triple-element electrostatic lens for focusing the electron beams and achieved a focused beam spot of 87 nm on the sample plane at the electron energy of 2 kV. High-angle coherent diffraction patterns of a suspended graphene sample corresponding to (0.62 Å){sup −1} were recorded. This workmore » demonstrated the potential of coherent diffractive imaging of thin two-dimensional materials, biological molecules, and nano-objects at a voltage between 1 and 10 kV. The ultimate goal of this instrument is to achieve atomic resolution of these materials with high contrast and little radiation damage.« less
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Digital Assays Part II: Digital Protein and Cell Assays.
Basu, Amar S
2017-08-01
A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.
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Towards advanced biological detection using surface enhanced raman scattering (SERS)-based sensors
NASA Astrophysics Data System (ADS)
Hankus, Mikella E.; Stratis-Cullum, Dimitra N.; Pellegrino, Paul M.
2010-08-01
The Army has a need for an accurate, fast, reliable and robust means to identify and quantify defense related materials. Raman spectroscopy is a form of vibrational spectroscopy that is rapidly becoming a valuable tool for homeland defense applications, as it is well suited for the molecular identification of a variety of compounds, including explosives and chemical and biological hazards. To measure trace levels of these types of materials, surface enhanced Raman scattering (SERS), a specialized form of Raman scattering, can be employed. The SERS enhancements are produced on, or in close proximity to, a nanoscale roughened metal surface and are typically associated with increased local electromagnetic field strengths. However, before application of SERS in the field and in particular to biological and other hazard sensing applications, significant improvements in substrate performance are needed. In this work, we will report the use of several SERS substrate architectures (colloids, film-over-nanospheres (FONs) and commercially available substrates) for detecting and differentiating numerous endospore samples. The variance in spectra as obtained using different sensing architectures will also be discussed. Additionally, the feasibility of using a modified substrate architecture that is tailored with molecular recognition probe system for detecting biological samples will be explored. We will discuss the progress towards an advanced, hybrid molecular recognition with a SERS/Fluorescence nanoprobe system including the optimization, fabrication, and spectroscopic analysis of samples on a commercially available substrate. Additionally, the feasibility of using this single-step switching architecture for hazard material detection will also be explored.
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Standoff detection of chemical and biological threats using laser-induced breakdown spectroscopy.
Gottfried, Jennifer L; De Lucia, Frank C; Munson, Chase A; Miziolek, Andrzej W
2008-04-01
Laser-induced breakdown spectroscopy (LIBS) is a promising technique for real-time chemical and biological warfare agent detection in the field. We have demonstrated the detection and discrimination of the biological warfare agent surrogates Bacillus subtilis (BG) (2% false negatives, 0% false positives) and ovalbumin (0% false negatives, 1% false positives) at 20 meters using standoff laser-induced breakdown spectroscopy (ST-LIBS) and linear correlation. Unknown interferent samples (not included in the model), samples on different substrates, and mixtures of BG and Arizona road dust have been classified with reasonable success using partial least squares discriminant analysis (PLS-DA). A few of the samples tested such as the soot (not included in the model) and the 25% BG:75% dust mixture resulted in a significant number of false positives or false negatives, respectively. Our preliminary results indicate that while LIBS is able to discriminate biomaterials with similar elemental compositions at standoff distances based on differences in key intensity ratios, further work is needed to reduce the number of false positives/negatives by refining the PLS-DA model to include a sufficient range of material classes and carefully selecting a detection threshold. In addition, we have demonstrated that LIBS can distinguish five different organophosphate nerve agent simulants at 20 meters, despite their similar stoichiometric formulas. Finally, a combined PLS-DA model for chemical, biological, and explosives detection using a single ST-LIBS sensor has been developed in order to demonstrate the potential of standoff LIBS for universal hazardous materials detection.
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3D imaging of cells and tissues by focused ion beam/scanning electron microscopy (FIB/SEM).
Drobne, Damjana
2013-01-01
Integration of a scanning electron microscope (SEM) and focused ion beam (FIB) technology into a single FIB/SEM system permits use of the FIB as a nano-scalpel to reveal site-specific subsurface microstructures which can be examined in great detail by SEM. The FIB/SEM technology is widely used in the semiconductor industry and material sciences, and recently its use in the life sciences has been initiated. Samples for FIB/SEM investigation can be either embedded in a plastic matrix, the traditional means of preparation of transmission electron microscopy (TEM) specimens, or simply dried as in samples prepared for SEM imaging. Currently, FIB/SEM is used in the life sciences for (a) preparation by the lift-out technique of lamella for TEM analysis, (b) tomography of samples embedded in a matrix, and (c) in situ site-specific FIB milling and SEM imaging using a wide range of magnifications. Site-specific milling and imaging has attracted wide interest as a technique in structural research of single eukaryotic and prokaryotic cells, small animals, and different animal tissue, but it still remains to be explored more thoroughly. In the past, preparation of samples for site-specific milling and imaging by FIB/SEM has typically adopted the embedding techniques used for TEM samples, and which have been very well described in the literature. Sample preparation protocols for the use of dried samples in FIB/SEM have been less well investigated. The aim of this chapter is to encourage application of FIB/SEM on dried biological samples. A detailed description of conventional dried sample preparation and FIB/SEM investigation of dried biological samples is presented. The important steps are described and illustrated, and direct comparison between embedded and dried samples of same tissues is provided. The ability to discover links between gross morphology of the tissue or organ, surface characteristics of any selected region, and intracellular structural details on the nanometer scale is an appealing application of electron microscopy in the life sciences and merits further exploration.
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High-throughput biological small-angle X-ray scattering with a robotically loaded capillary cell
Nielsen, S. S.; Møller, M.; Gillilan, R. E.
2012-01-01
With the rise in popularity of biological small-angle X-ray scattering (BioSAXS) measurements, synchrotron beamlines are confronted with an ever-increasing number of samples from a wide range of solution conditions. To meet these demands, an increasing number of beamlines worldwide have begun to provide automated liquid-handling systems for sample loading. This article presents an automated sample-loading system for BioSAXS beamlines, which combines single-channel disposable-tip pipetting with a vacuum-enclosed temperature-controlled capillary flow cell. The design incorporates an easily changeable capillary to reduce the incidence of X-ray window fouling and cross contamination. Both the robot-control and the data-processing systems are written in Python. The data-processing code, RAW, has been enhanced with several new features to form a user-friendly BioSAXS pipeline for the robot. The flow cell also supports efficient manual loading and sample recovery. An effective rinse protocol for the sample cell is developed and tested. Fluid dynamics within the sample capillary reveals a vortex ring pattern of circulation that redistributes radiation-damaged material. Radiation damage is most severe in the boundary layer near the capillary surface. At typical flow speeds, capillaries below 2 mm in diameter are beginning to enter the Stokes (creeping flow) regime in which mixing due to oscillation is limited. Analysis within this regime shows that single-pass exposure and multiple-pass exposure of a sample plug are functionally the same with regard to exposed volume when plug motion reversal is slow. The robot was tested on three different beamlines at the Cornell High-Energy Synchrotron Source, with a variety of detectors and beam characteristics, and it has been used successfully in several published studies as well as in two introductory short courses on basic BioSAXS methods. PMID:22509071
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Quantitative biology of single neurons
Eberwine, James; Lovatt, Ditte; Buckley, Peter; Dueck, Hannah; Francis, Chantal; Kim, Tae Kyung; Lee, Jaehee; Lee, Miler; Miyashiro, Kevin; Morris, Jacqueline; Peritz, Tiina; Schochet, Terri; Spaethling, Jennifer; Sul, Jai-Yoon; Kim, Junhyong
2012-01-01
The building blocks of complex biological systems are single cells. Fundamental insights gained from single-cell analysis promise to provide the framework for understanding normal biological systems development as well as the limits on systems/cellular ability to respond to disease. The interplay of cells to create functional systems is not well understood. Until recently, the study of single cells has concentrated primarily on morphological and physiological characterization. With the application of new highly sensitive molecular and genomic technologies, the quantitative biochemistry of single cells is now accessible. PMID:22915636
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Discovering Physical Samples Through Identifiers, Metadata, and Brokering
NASA Astrophysics Data System (ADS)
Arctur, D. K.; Hills, D. J.; Jenkyns, R.
2015-12-01
Physical samples, particularly in the geosciences, are key to understanding the Earth system, its history, and its evolution. Our record of the Earth as captured by physical samples is difficult to explain and mine for understanding, due to incomplete, disconnected, and evolving metadata content. This is further complicated by differing ways of classifying, cataloguing, publishing, and searching the metadata, especially when specimens do not fit neatly into a single domain—for example, fossils cross disciplinary boundaries (mineral and biological). Sometimes even the fundamental classification systems evolve, such as the geological time scale, triggering daunting processes to update existing specimen databases. Increasingly, we need to consider ways of leveraging permanent, unique identifiers, as well as advancements in metadata publishing that link digital records with physical samples in a robust, adaptive way. An NSF EarthCube Research Coordination Network (RCN) called the Internet of Samples (iSamples) is now working to bridge the metadata schemas for biological and geological domains. We are leveraging the International Geo Sample Number (IGSN) that provides a versatile system of registering physical samples, and working to harmonize this with the DataCite schema for Digital Object Identifiers (DOI). A brokering approach for linking disparate catalogues and classification systems could help scale discovery and access to the many large collections now being managed (sometimes millions of specimens per collection). This presentation is about our community building efforts, research directions, and insights to date.
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Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John
2016-11-01
Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.
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Mamontov, Eugene
2016-06-29
We present a concept and ray-tracing simulation results of a mechanical device that will enable inelastic neutron scattering measurements where the data at energy transfers from a few eV to several hundred meV can be collected in a single, gapless spectrum. Besides covering 5 orders of magnitude on the energy (time) scale, the device provides data over 2 orders of magnitude on the scattering momentum (length) scale in a single measurement. Such capabilities are geared primarily toward soft and biological matter, where the broad dynamical features of relaxation origin largely overlap with vibration features, thus necessitating gapless spectral coverage overmore » several orders of magnitude in time and space. Furthermore, neutron scattering experiments with such a device are performed with a fixed neutron final energy, which enables measurements, with neutron energy loss in the sample, at arbitrarily low temperatures over the same broad spectral range. Lastly, this capability is also invaluable in biological and soft matter research, as the variable temperature dependence of different relaxation components allows their separation in the scattering spectra as a function of temperature.« less
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Genetic Association of MPPED2 and ACTN2 with Dental Caries
Stanley, B.O.C.; Feingold, E.; Cooper, M.; Vanyukov, M.M.; Maher, B.S.; Slayton, R.L.; Willing, M.C.; Reis, S.E.; McNeil, D.W.; Crout, R.J.; Weyant, R.J.; Levy, S.M.; Vieira, A.R.; Marazita, M.L.; Shaffer, J.R.
2014-01-01
The first genome-wide association study of dental caries focused on primary teeth in children aged 3 to 12 yr and nominated several novel genes: ACTN2, EDARADD, EPHA7, LPO, MPPED2, MTR, and ZMPSTE24. Here we interrogated 156 single-nucleotide polymorphisms (SNPs) within these candidate genes for evidence of association with dental caries experience in 13 race- and age-stratified samples from 6 independent studies (n = 3600). Analysis was performed separately for each sample, and results were combined across samples via meta-analysis. MPPED2 was significantly associated with caries via meta-analysis across the 5 childhood samples, with 4 SNPs showing significant associations after gene-wise adjustment for multiple comparisons (p < .0026). These results corroborate the previous genome-wide association study, although the functional role of MPPED2 in caries etiology remains unknown. ACTN2 also showed significant association via meta-analysis across childhood samples (p = .0014). Moreover, in adults, genetic association was observed for ACTN2 SNPs in individual samples (p < .0025), but no single SNP was significant via meta-analysis across all 8 adult samples. Given its compelling biological role in organizing ameloblasts during amelogenesis, this study strengthens the hypothesis that ACTN2 influences caries risk. Results for the other candidate genes neither proved nor precluded their associations with dental caries. PMID:24810274
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang, Yijia; Zeinhom, Mohamed M. A.; Yang, Mingming
Onsite rapid detection of herbicide and herbicide residuals in environmental and biological specimens is important for agriculture, environment, food safety, and health care. Traditional method for herbicide detection requires expensive laboratory equipment and a long turn-round time. In this work, we developed a single-stripe microliter plate smartphone colorimetric device for rapid and low-cost in-field test. This portable smartphone platform is capable of screening 8 samples in a microplate single-stripe. The device combined the advantages of small size (50×100×160 mm3) and low cost ($10). The platform was calibrated by using two different dye solutions, i.e. methyl blue (MB) and Rhodamine B,more » for green and red channels. The results showed good correlation with results attained from a traditional laboratory reader. We demonstrated the application of this platform for an herbicide, 2,4-Dichlorophenoxyacetic acid detection in the range of 1 ppb to 80 ppb. Spiked samples of tap water, rat serum, plasma and human serum were tested by our device. Recoveries obtained varied from 95.6% to 105.2% for all spiked samples using the microplate reader and from 93.7% to 106.9% using the smartphone device. This work validated that the smartphone optical sensing platform is comparable to the commercial microplate reader, it is eligible for onsite rapid and low-cost detection of herbicide for environmental evaluation and biological monitoring.« less
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Searching for Life on Mars: Selection of Molecular Targets for ESA's Aurora ExoMars Mission
NASA Astrophysics Data System (ADS)
Parnell, John; Cullen, David; Sims, Mark R.; Bowden, Stephen; Cockell, Charles S.; Court, Richard; Ehrenfreund, Pascale; Gaubert, Francois; Grant, William; Parro, Victor; Rohmer, Michel; Sephton, Mark; Stan-Lotter, Helga; Steele, Andrew; Toporski, Jan; Vago, Jorge
2007-08-01
The European Space Agency's ExoMars mission will seek evidence of organic compounds of biological and non-biological origin at the martian surface. One of the instruments in the Pasteur payload may be a Life Marker Chip that utilizes an immunoassay approach to detect specific organic molecules or classes of molecules. Therefore, it is necessary to define and prioritize specific molecular targets for antibody development. Target compounds have been selected to represent meteoritic input, fossil organic matter, extant (living, recently dead) organic matter, and contamination. Once organic molecules are detected on Mars, further information is likely to derive from the detailed distribution of compounds rather than from single molecular identification. This will include concentration gradients beneath the surface and gradients from generic to specific compounds. The choice of biomarkers is informed by terrestrial biology but is wide ranging, and nonterrestrial biology may be evident from unexpected molecular distributions. One of the most important requirements is to sample where irradiation and oxidation are minimized, either by drilling or by using naturally excavated exposures. Analyzing regolith samples will allow for the search of both extant and fossil biomarkers, but sequential extraction would be required to optimize the analysis of each of these in turn.
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Searching for life on Mars: selection of molecular targets for ESA's aurora ExoMars mission.
Parnell, John; Cullen, David; Sims, Mark R; Bowden, Stephen; Cockell, Charles S; Court, Richard; Ehrenfreund, Pascale; Gaubert, Francois; Grant, William; Parro, Victor; Rohmer, Michel; Sephton, Mark; Stan-Lotter, Helga; Steele, Andrew; Toporski, Jan; Vago, Jorge
2007-08-01
The European Space Agency's ExoMars mission will seek evidence of organic compounds of biological and non-biological origin at the martian surface. One of the instruments in the Pasteur payload may be a Life Marker Chip that utilizes an immunoassay approach to detect specific organic molecules or classes of molecules. Therefore, it is necessary to define and prioritize specific molecular targets for antibody development. Target compounds have been selected to represent meteoritic input, fossil organic matter, extant (living, recently dead) organic matter, and contamination. Once organic molecules are detected on Mars, further information is likely to derive from the detailed distribution of compounds rather than from single molecular identification. This will include concentration gradients beneath the surface and gradients from generic to specific compounds. The choice of biomarkers is informed by terrestrial biology but is wide ranging, and nonterrestrial biology may be evident from unexpected molecular distributions. One of the most important requirements is to sample where irradiation and oxidation are minimized, either by drilling or by using naturally excavated exposures. Analyzing regolith samples will allow for the search of both extant and fossil biomarkers, but sequential extraction would be required to optimize the analysis of each of these in turn.
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1988-11-01
Bilayer ........................................... 14 5. Current-Voltage Curve for Gramacidin in a Lecithin -Sphingomyelin Patch Bilayer... lecithin (Avanti). 9 2. MATERIALS 2.1 Patch Microprobe Instrumentation. The basis of the microprobe system is an AxoPatch Patch- Clamping Amplifier System...histogram of 1024 events cut above 2 pA. Events sampled are thought to be from the same single gramacidin channel in a lecithin : sphingomyelin (5:1) patch
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Viscoelastic characterization of soft biological materials
NASA Astrophysics Data System (ADS)
Nayar, Vinod Timothy
Progressive and irreversible retinal diseases are among the primary causes of blindness in the United States, attacking the cells in the eye that transform environmental light into neural signals for the optic pathway. Medical implants designed to restore visual function to afflicted patients can cause mechanical stress and ultimately damage to the host tissues. Research shows that an accurate understanding of the mechanical properties of the biological tissues can reduce damage and lead to designs with improved safety and efficacy. Prior studies on the mechanical properties of biological tissues show characterization of these materials can be affected by environmental, length-scale, time, mounting, stiffness, size, viscoelastic, and methodological conditions. Using porcine sclera tissue, the effects of environmental, time, and mounting conditions are evaluated when using nanoindentation. Quasi-static tests are used to measure reduced modulus during extended exposure to phosphate-buffered saline (PBS), as well as the chemical and mechanical analysis of mounting the sample to a solid substrate using cyanoacrylate. The less destructive nature of nanoindentation tests allows for variance of tests within a single sample to be compared to the variance between samples. The results indicate that the environmental, time, and mounting conditions can be controlled for using modified nanoindentation procedures for biological samples and are in line with averages modulus values from previous studies but with increased precision. By using the quasi-static and dynamic characterization capabilities of the nanoindentation setup, the additional stiffness and viscoelastic variables are measured. Different quasi-static control methods were evaluated along with maximum load parameters and produced no significant difference in reported reduced modulus values. Dynamic characterization tests varied frequency and quasi-static load, showing that the agar could be modeled as a linearly elastic material. The effects of sample stiffness were evaluated by testing both the quasi-static and dynamic mechanical properties of different concentration agar samples, ranging from 0.5% to 5.0%. The dynamic nanoindentation protocol showed some sensitivity to sample stiffness, but characterization remained consistently applicable to soft biological materials. Comparative experiments were performed on both 0.5% and 5.0% agar as well as porcine eye tissue samples using published dynamic macrocompression standards. By comparing these new tests to those obtained with nanoindentation, the effects due to length-scale, stiffness, size, viscoelastic, and methodological conditions are evaluated. Both testing methodologies can be adapted for the environmental and mounting conditions, but the limitations of standardized macro-scale tests are explored. The factors affecting mechanical characterization of soft and thin viscoelastic biological materials are researched and a comprehensive protocol is presented. This work produces material mechanical properties for use in improving future medical implant designs on a wide variety of biological tissue and materials.
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NASA Astrophysics Data System (ADS)
Ujiie, Y.; Kimoto, K.; Ishimura, T.
2017-12-01
Planktonic foraminifers are widely used in the studies of paleontology and paleoceanography, because the morphology of their calcareous shells is enough highly variable to identify the morphospecies and the chemical composition of the shells reflect ambient seawater condition. Although the morphospecies were believed to represent environments associating with latitudinal temperature range of the world ocean, molecular phylogeographic studies have unveiled the presence of multiple biological species in a single morphospecies and their species-specific distributions. This implicates the actual complexity of planktonic foraminiferal ecology. Conversely, these biological species have a high potential for providing novel ecological and environmental information to us. In order to reassess the morphological and geochemical characters of biological species, the DNA extraction method with the guanidium isothiocyanate buffer was developed to preserve the calcareous shells. The present study carefully tested the physical and chemical damages of the DNA extraction process to the shells, by our novel approaches with geochemical analysis of the shells after non-destructive analysis for morphometrics on a same specimen. First, we checked the changes of the shell densities between pre- and post-DNA extraction by using the micro-focus X-ray CT (MXCT) scanning. Based on the simultaneous measurement of a sample and the standard material, we confirmed no significant changes to the shell densities through the DNA extraction process. As a next step, we compared stable oxygen and carbon isotopes among individuals of three sample sets: (1) no chemical and incubation as control, (2) incubation in the DNA extraction buffer at 65-70°C for 40 minutes as standard way, and (3) incubation in the DNA extraction buffer at 65-70°C for 120 minutes, by using the microscale isotopic analytical system (MICAL3c). Consequently, there were no significant differences among the three sample sets. These examinations clearly certified that we define morphological and geochemical features from same specimens after genetic identification. Thus, our novel approach (SAMSI) provides future studies to establish the accurate ecological and environmental proxies both in the modern and past oceans.
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Single-cell regulome data analysis by SCRAT.
Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai
2017-09-15
Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.
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Barteneva, Natasha S; Vorobjev, Ivan A
2018-01-01
In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.
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Database Resources of the BIG Data Center in 2018.
2018-01-04
The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Antibiotic Resistance and the Biology of History.
Landecker, Hannah
2016-12-01
Beginning in the 1940s, mass production of antibiotics involved the industrial-scale growth of microorganisms to harvest their metabolic products. Unfortunately, the use of antibiotics selects for resistance at answering scale. The turn to the study of antibiotic resistance in microbiology and medicine is examined, focusing on the realization that individual therapies targeted at single pathogens in individual bodies are environmental events affecting bacterial evolution far beyond bodies. In turning to biological manifestations of antibiotic use, sciences fathom material outcomes of their own previous concepts. Archival work with stored soil and clinical samples produces a record described here as 'the biology of history': the physical registration of human history in bacterial life. This account thus foregrounds the importance of understanding both the materiality of history and the historicity of matter in theories and concepts of life today.
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Imaging modes of atomic force microscopy for application in molecular and cell biology.
Dufrêne, Yves F; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas; Gerber, Christoph; Müller, Daniel J
2017-04-06
Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.
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Antibiotic Resistance and the Biology of History
2015-01-01
Beginning in the 1940s, mass production of antibiotics involved the industrial-scale growth of microorganisms to harvest their metabolic products. Unfortunately, the use of antibiotics selects for resistance at answering scale. The turn to the study of antibiotic resistance in microbiology and medicine is examined, focusing on the realization that individual therapies targeted at single pathogens in individual bodies are environmental events affecting bacterial evolution far beyond bodies. In turning to biological manifestations of antibiotic use, sciences fathom material outcomes of their own previous concepts. Archival work with stored soil and clinical samples produces a record described here as ‘the biology of history’: the physical registration of human history in bacterial life. This account thus foregrounds the importance of understanding both the materiality of history and the historicity of matter in theories and concepts of life today. PMID:28458609
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How cryo‐electron microscopy and X‐ray crystallography complement each other
Wang, Jia‐Wei
2016-01-01
Abstract With the ability to resolve structures of macromolecules at atomic resolution, X‐ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo‐EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X‐ray crystallography and cryo‐EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms. PMID:27543495
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How cryo-electron microscopy and X-ray crystallography complement each other.
Wang, Hong-Wei; Wang, Jia-Wei
2017-01-01
With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo-EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms. © 2016 The Protein Society.
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NASA Astrophysics Data System (ADS)
Loubere, Paul; Fariduddin, Mohammad
1999-03-01
We present a quantitative method, based on the relative abundances of benthic foraminifera in deep-sea sediments, for estimating surface ocean biological productivity over the timescale of centuries to millennia. We calibrate the method using a global data set composed of 207 samples from the Atlantic, Pacific, and Indian Oceans from a water depth range between 2300 and 3600 m. The sample set was developed so that other, potentially significant, environmental variables would be uncorrelated to overlying surface ocean productivity. A regression of assemblages against productivity yielded an r2 = 0.89 demonstrating a strong productivity signal in the faunal data. In addition, we examined assemblage response to annual variability in biological productivity (seasonality). Our data set included a range of seasonalities which we quantified into a seasonality index using the pigment color bands from the coastal zone color scanner (CZCS). The response of benthic foraminiferal assemblage composition to our seasonality index was tested with regression analysis. We obtained a statistically highly significant r2 = 0.75. Further, discriminant function analysis revealed a clear separation among sample groups based on surface ocean productivity and our seasonality index. Finally, we tested the response of benthic foraminiferal assemblages to three different modes of seasonality. We observed a distinct separation of our samples into groups representing low seasonal variability, strong seasonality with a single main productivity event in the year, and strong seasonality with multiple productivity events in the year. Reconstructing surface ocean biological productivity with benthic foraminifera will aid in modeling marine biogeochemical cycles. Also, estimating mode and range of annual seasonality will provide insight to changing oceanic processes, allowing the examination of the mechanisms causing changes in the marine biotic system over time. This article contains supplementary material.
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Integrative Exploratory Analysis of Two or More Genomic Datasets.
Meng, Chen; Culhane, Aedin
2016-01-01
Exploratory analysis is an essential step in the analysis of high throughput data. Multivariate approaches such as correspondence analysis (CA), principal component analysis, and multidimensional scaling are widely used in the exploratory analysis of single dataset. Modern biological studies often assay multiple types of biological molecules (e.g., mRNA, protein, phosphoproteins) on a same set of biological samples, thereby creating multiple different types of omics data or multiassay data. Integrative exploratory analysis of these multiple omics data is required to leverage the potential of multiple omics studies. In this chapter, we describe the application of co-inertia analysis (CIA; for analyzing two datasets) and multiple co-inertia analysis (MCIA; for three or more datasets) to address this problem. These methods are powerful yet simple multivariate approaches that represent samples using a lower number of variables, allowing a more easily identification of the correlated structure in and between multiple high dimensional datasets. Graphical representations can be employed to this purpose. In addition, the methods simultaneously project samples and variables (genes, proteins) onto the same lower dimensional space, so the most variant variables from each dataset can be selected and associated with samples, which can be further used to facilitate biological interpretation and pathway analysis. We applied CIA to explore the concordance between mRNA and protein expression in a panel of 60 tumor cell lines from the National Cancer Institute. In the same 60 cell lines, we used MCIA to perform a cross-platform comparison of mRNA gene expression profiles obtained on four different microarray platforms. Last, as an example of integrative analysis of multiassay or multi-omics data we analyzed transcriptomic, proteomic, and phosphoproteomic data from pluripotent (iPS) and embryonic stem (ES) cell lines.
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SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY
DOE Office of Scientific and Technical Information (OSTI.GOV)
Professor William Moerner
2010-07-09
The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together,more » and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in Tuscany, provides an avenue for scientists from different disciplines to interact and brainstorm and promotes cross-disciplinary collaborations directed toward compelling biological problems.« less
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NASA Astrophysics Data System (ADS)
Srivastava, Vishal; Mehta, D. S.
2013-02-01
To quantitatively obtain the phase map of Onion and human red blood cell (RBC) from white light interferogram we used Hilbert transform color fringe analysis technique. The three Red, Blue and Green color components are decomposed from single white light interferogram and Refractive index profile for Red, Blue and Green colour were computed in a completely non-invasive manner for Onion and human RBC. The present technique might be useful for non-invasive determination of the refractive index variation within cells and tissues and morphological features of sample with ease of operation and low cost.
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Single-cell sequencing technologies: current and future.
Liang, Jialong; Cai, Wanshi; Sun, Zhongsheng
2014-10-20
Intensively developed in the last few years, single-cell sequencing technologies now present numerous advantages over traditional sequencing methods for solving the problems of biological heterogeneity and low quantities of available biological materials. The application of single-cell sequencing technologies has profoundly changed our understanding of a series of biological phenomena, including gene transcription, embryo development, and carcinogenesis. However, before single-cell sequencing technologies can be used extensively, researchers face the serious challenge of overcoming inherent issues of high amplification bias, low accuracy and reproducibility. Here, we simply summarize the techniques used for single-cell isolation, and review the current technologies used in single-cell genomic, transcriptomic, and epigenomic sequencing. We discuss the merits, defects, and scope of application of single-cell sequencing technologies and then speculate on the direction of future developments. Copyright © 2014 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.
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Owen, R J; Heyes, C D; Knebel, D; Röcker, C; Nienhaus, G U
2006-07-01
In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution. (c) 2006 Wiley Periodicals, Inc.
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Cooperative Robotics and the Search for Extraterrestrial Life
NASA Technical Reports Server (NTRS)
Lupisella, M. L.
2000-01-01
If we think tenuous abodes of life may be hiding in remote extraterrestrial environmental niches, and if we want to assess the biological status of a given locale or entire planet before sending humans (perhaps because of contamination concerns or other motivations) then we face the challenge of robotically exploring a large space efficiently and in enough detail to have confidence in our assessment of the biological status of the environment in question. On our present schedule of perhaps two or so missions per opportunity, we will likely need a different exploratory approach than singular stationary landers or singular rover missions or sample return, because there appear to be fundamental limitations in these mission profiles to obtain the many samples we will likely need if we want to have confidence in assessing the biological status of an environment in which life could be hiding in remote environmental niches. Singular rover missions can potentially accommodate sampling over a fairly large area, but are still limited by range and can be a single point of failure. More importantly, such mission profiles have limited payload capabilities which are unlikely to meet the demanding requirements of life-detection. Sample return has the advantage of allowing sophisticated analysis of the sample, but also has the severe limitations associated with only being able to bring back a few samples. This presentation will suggest two cooperative robotic approaches for exploration that have the potential to overcome these difficulties and facilitate efficient and thorough life-detecting exploration of a large space. Given the two premises stated above, it appears at least two fundamental challenges have to be met simultaneously: (1) coverage of a large space and (2) bringing to bear a sophisticated suite of detection and experimental payloads on any specific location in order to address a major challenge in looking for extraterrestrial life: namely, executing a wide variety of detection scenarios and in situ experiments in order to gather the required data for a confident assessment that life has been detected and to, more generally, cover a wide range of extraterrestrial life possibilities. Cooperative robotics lends itself to this kind of problem because cooperation among the combined capabilities of a variety of simple single function agents can give rise to fairly complex task execution such as the search for and detection of extraterrestrial life.
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Cooperative Robotics and the Search for Extraterrestrial Life
NASA Technical Reports Server (NTRS)
Lupisella, Mark L.
2000-01-01
If we think tenuous abodes of life may be hiding in remote extraterrestrial environmental niches, and if we want to assess the biological status of a given locale or entire planet before sending humans (perhaps because of contamination concerns or other motivations) then we face the challenge of robotically exploring a large space efficiently and in enough detail to have confidence in our assessment of the biological status of the environment in question. On our present schedule of perhaps two or so missions per opportunity, we will likely need a different exploratory approach than singular stationary landers or singular rover missions or sample return, because there appear to be fundamental limitations in these mission profiles to-obtain the many samples we will likely need if we want to have confidence in assessing the biological status of an environment in which life could be hiding in remote environmental niches. Singular rover missions can potentially accommodate sampling over a fairly large area, but are still limited by range and can be a single point of failure. More importantly, such mission profiles have limited payload capabilities which are unlikely to meet the demanding requirements of life-detection. Sample return has the advantage of allowing sophisticated analysis of the sample, but also has the severe limitations associated with only being able to bring back a few samples. This presentation will suggest two cooperative robotic approaches for exploration that have the potential to overcome these difficulties and facilitate efficient and thorough life-detecting exploration of a large space. Given the two premises state above, it appears at least two fundamental challenges have to be met simultaneously: coverage of a large space and bringing to bear a sophisticated suite of detection and experimental payloads on any specific location in order to address a major challenge in looking for extraterrestrial life: namely, executing a wide variety of detection scenarios and in situ experiments in order to gather the required data for a confident assessment that life has been detected and to, more generally, cover a wide range of extraterrestrial life possibilities. Cooperative robotics ]ends itself to this kind of problem because cooperation among the combined capabilities of a variety of simple single function agents can give rise to fairly complex task execution such as the search for and detection of extraterrestrial life.
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Family Structure, Transitions and Psychiatric Disorders Among Puerto Rican Children
Santesteban-Echarri, Olga; Eisenberg, Ruth E.; Bird, Hector R.; Canino, Glorisa J.; Duarte, Cristiane S.
2016-01-01
This paper examines whether family structure and its transitions are associated with internalizing and externalizing psychiatric disorders among Puerto Rican-origin children. It uses longitudinal data (three waves) from the Boricua Youth Study, which includes probability samples of children in the South Bronx (New York) and San Juan (Puerto Rico) (n = 2,142). We also examine factors which may explain how family structure and transitions may be related to child psychiatric disorders. Our results show that for both internalizing and externalizing disorders there were no significant differences between children of cohabiting (biological or step) parents or of single parents compared to children of married biological parents. In Puerto Rico only, transitioning once from a two-parent family to a single-parent family was related to child internalizing disorders. Family transitions were not associated with externalizing disorders at either site. Context may be an important factor shaping the risk that family dissolution is followed by an internalizing disorder among children. PMID:28713212
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Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq.
Chen, Jun; Suo, Shengbao; Tam, Patrick Pl; Han, Jing-Dong J; Peng, Guangdun; Jing, Naihe
2017-03-01
Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously. The Geo-seq protocol allows the profiling of transcriptome information from only a small number cells and retains their native spatial information. This protocol has wide potential applications to address biological and pathological questions of cellular properties such as prospective cell fates, biological function and the gene regulatory network. Geo-seq has been applied to investigate the spatial transcriptome of mouse early embryo, mouse brain, and pathological liver and sperm tissues. The entire protocol from tissue collection and microdissection to sequencing requires ∼5 d, Data analysis takes another 1 or 2 weeks, depending on the amount of data and the speed of the processor.
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Cappitelli, Francesca; Principi, Pamela; Pedrazzani, Roberta; Toniolo, Lucia; Sorlini, Claudia
2007-10-15
Surfaces are continuously exposed to physical, chemical and biological degradation. Among the biological agents that cause deterioration, microorganisms are of critical importance. This work is part of a research programme for the characterisation of the alterations of the Milan Cathedral (Italy). Four stone samples of the Milan Cathedral were chemically analysed and the microbiological growth assessed. X-ray diffraction (XRD) showed that calcite was always present in each sample and one sample was also characterised by the chemical form of alteration gypsum. Using Fourier Transform Infrared Spectroscopy (FTIR) together with Scanning Electron Microscopy (SEM), it was possible to prove that the samples were consolidated with the synthetic acrylics and epoxy resins. The green-black biological patinas of the specimens were studied using cultivation, microscope observations and a method for single-cell detection. Sampling for fluorescent in-situ hybridisation (FISH), with ribosomal RNA targeted oligonucleotide probes, was also performed using adhesive tapes. The bulk of the prokaryotes were Bacteria but some Archaea were also found. The bacterial cells were further characterised using specific probes for Cyanobacteria, and alpha-, beta-and gamma-Proteobacteria. In addition, black fungi isolated from the stone and the fungi of the standard ASTM G21-96(2002) method were employed to test if the detected synthetic resins could be used as the sole source of carbon and energy. One isolated Cladosporium sp. attacked the freshly dried acrylic resin. Results show that the detected bacteria and fungi can cause severe damage both to the stone monument and its synthetic consolidants.
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Wang, Jiaojiao; Lan, Jingfeng; Li, Huihui; Liu, Xiaoyan; Zhang, Haixia
2017-01-01
In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH-sensitive functional mesoporous silica materials composed of 2-(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH-sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five-protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum albumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms. Copyright © 2016 Elsevier B.V. All rights reserved.
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Mapping a multiplexed zoo of mRNA expression.
Choi, Harry M T; Calvert, Colby R; Husain, Naeem; Huss, David; Barsi, Julius C; Deverman, Benjamin E; Hunter, Ryan C; Kato, Mihoko; Lee, S Melanie; Abelin, Anna C T; Rosenthal, Adam Z; Akbari, Omar S; Li, Yuwei; Hay, Bruce A; Sternberg, Paul W; Patterson, Paul H; Davidson, Eric H; Mazmanian, Sarkis K; Prober, David A; van de Rijn, Matt; Leadbetter, Jared R; Newman, Dianne K; Readhead, Carol; Bronner, Marianne E; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E; Pierce, Niles A
2016-10-01
In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. © 2016. Published by The Company of Biologists Ltd.
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Mapping a multiplexed zoo of mRNA expression
Choi, Harry M. T.; Calvert, Colby R.; Husain, Naeem; Huss, David; Barsi, Julius C.; Deverman, Benjamin E.; Hunter, Ryan C.; Kato, Mihoko; Lee, S. Melanie; Abelin, Anna C. T.; Rosenthal, Adam Z.; Akbari, Omar S.; Li, Yuwei; Hay, Bruce A.; Sternberg, Paul W.; Patterson, Paul H.; Davidson, Eric H.; Mazmanian, Sarkis K.; Prober, David A.; van de Rijn, Matt; Leadbetter, Jared R.; Newman, Dianne K.; Readhead, Carol; Bronner, Marianne E.; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E.
2016-01-01
In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. PMID:27702788
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Uludağ, Yildiz; Piletsky, Sergey A; Turner, Anthony P F; Cooper, Matthew A
2007-11-01
Biomimetic recognition elements employed for the detection of analytes are commonly based on proteinaceous affibodies, immunoglobulins, single-chain and single-domain antibody fragments or aptamers. The alternative supra-molecular approach using a molecularly imprinted polymer now has proven utility in numerous applications ranging from liquid chromatography to bioassays. Despite inherent advantages compared with biochemical/biological recognition (which include robustness, storage endurance and lower costs) there are few contributions that describe quantitative analytical applications of molecularly imprinted polymers for relevant small molecular mass compounds in real-world samples. There is, however, significant literature describing the use of low-power, portable piezoelectric transducers to detect analytes in environmental monitoring and other application areas. Here we review the combination of molecularly imprinted polymers as recognition elements with piezoelectric biosensors for quantitative detection of small molecules. Analytes are classified by type and sample matrix presentation and various molecularly imprinted polymer synthetic fabrication strategies are also reviewed.
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Solid phase sequencing of biopolymers
Cantor, Charles; Koster, Hubert
2010-09-28
This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.
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MARK-AGE standard operating procedures (SOPs): A successful effort.
Moreno-Villanueva, María; Capri, Miriam; Breusing, Nicolle; Siepelmeyer, Anne; Sevini, Federica; Ghezzo, Alessandro; de Craen, Anton J M; Hervonen, Antti; Hurme, Mikko; Schön, Christiane; Grune, Tilman; Franceschi, Claudio; Bürkle, Alexander
2015-11-01
Within the MARK-AGE project, a population study (3337 subjects) was conducted to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any single marker. The MARK-AGE project involves 14 European countries and a total of 26 research centres. In such a study, standard operating procedures (SOPs) are an essential task, which are binding for all MARK-AGE Beneficiaries. The SOPs cover all aspects of subject's recruitment, collection, shipment and distribution of biological samples (blood and its components, buccal mucosa cells or BMC and urine) as well as the anthropometric measurements and questionnaires. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Dehomogenized Elastic Properties of Heterogeneous Layered Materials in AFM Indentation Experiments.
Lee, Jia-Jye; Rao, Satish; Kaushik, Gaurav; Azeloglu, Evren U; Costa, Kevin D
2018-06-05
Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at submicron length scales. However, such samples are often structurally heterogeneous even at the local level, with different regions having distinct mechanical properties. Physical or chemical disruption can isolate individual structural elements but may alter the properties being measured. Therefore, to determine the micromechanical properties of intact heterogeneous multilayered samples indented by AFM, we propose the Hybrid Eshelby Decomposition (HED) analysis, which combines a modified homogenization theory and finite element modeling to extract layer-specific elastic moduli of composite structures from single indentations, utilizing knowledge of the component distribution to achieve solution uniqueness. Using finite element model-simulated indentation of layered samples with micron-scale thickness dimensions, biologically relevant elastic properties for incompressible soft tissues, and layer-specific heterogeneity of an order of magnitude or less, HED analysis recovered the prescribed modulus values typically within 10% error. Experimental validation using bilayer spin-coated polydimethylsiloxane samples also yielded self-consistent layer-specific modulus values whether arranged as stiff layer on soft substrate or soft layer on stiff substrate. We further examined a biophysical application by characterizing layer-specific microelastic properties of full-thickness mouse aortic wall tissue, demonstrating that the HED-extracted modulus of the tunica media was more than fivefold stiffer than the intima and not significantly different from direct indentation of exposed media tissue. Our results show that the elastic properties of surface and subsurface layers of microscale synthetic and biological samples can be simultaneously extracted from the composite material response to AFM indentation. HED analysis offers a robust approach to studying regional micromechanics of heterogeneous multilayered samples without destructively separating individual components before testing. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V
2015-03-04
Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.
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Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH.
Bolková, Jitka; Lanctôt, Christian
2016-04-01
Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism. Copyright © 2015 Elsevier Inc. All rights reserved.
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Biological Information Processing in Single Microtubules
2014-03-05
single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 3. Paul Davies Beyond Center at Arizona State University...Phoenix) Phoenix, workshop on quantum biology and cancer research, Experimental studies on single microtubule, 25-27 October 2010, Tempe, Arizona...State University, USA 4. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in microtubule, Towards a
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Single-cell proteomics: potential implications for cancer diagnostics.
Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T
2016-01-01
Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.
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Daaboul, George G; Lopez, Carlos A; Chinnala, Jyothsna; Goldberg, Bennett B; Connor, John H; Ünlü, M Selim
2014-06-24
Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.
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Dridi, M; Rosseel, T; Orton, R; Johnson, P; Lecollinet, S; Muylkens, B; Lambrecht, B; Van Borm, S
2015-10-01
West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 × in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Dmitriev, A K; Konovalov, A N; Ul'yanov, V A
2015-12-31
The autodyne signal arising in an Er fibre laser in the course of evaporating biological models of different types is studied and the possibility of recognising the biotissue type using the method of autodyne detection of the backscattered Doppler signal is assessed. In the experiments we modelled the process of surgical intervention using the contact (hole perforation with the Er laser fibre) and noncontact (surface evaporation with the focused radiation) regimes of impact on different biological models. The amplitude – frequency characteristic of the autodyne detection for the Er fibre laser is measured and the initial spectra of the backscatteredmore » Doppler signal arising under the action of laser radiation on the samples of biological models are obtained. The experiments have shown that the spectra of the backscattered Doppler signal, arising in the course of the contact and noncontact action of the Er fibre laser on different biological models, demonstrate clear-cut distinctions. (control of laser radiation parameters)« less
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Afornali, Alessandro; Vecchi, Rodrigo de; Stuart, Rodrigo Makowiecky; Dieamant, Gustavo; Oliveira, Luciana Lima de; Brohem, Carla Abdo; Feferman, Israel Henrique Stokfisz; Fabrício, Lincoln Helder Zambaldi; Lorencini, Márcio
2013-01-01
The sum of environmental and genetic factors affects the appearance and function of the skin as it ages. The identification of molecular changes that take place during skin aging provides biomarkers and possible targets for therapeutic intervention. Retinoic acid in different formulations has emerged as an alternative to prevent and repair age-related skin damage. To understand the effects of different retinoid formulations on the expression of genes associated with biological processes that undergo changes during skin aging. Ex-vivo skin samples were treated topically with different retinoid formulations. The modulation of biological processes associated with skin aging was measured by Reverse Transcription quantitative PCR (RT-qPCR). A formulation containing microencapsulated retinol and a blend of active ingredients prepared as a triple nanoemulsion provided the best results for the modulation of biological, process-related genes that are usually affected during skin aging. This association proved to be therapeutically more effective than tretinoin or microencapsulated retinol used singly.
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NanoSIMS for biological applications: Current practices and analyses
Nunez, Jamie R.; Renslow, Ryan S.; Cliff, III, John B.; ...
2017-09-27
Secondary ion mass spectrometry (SIMS) has become an increasingly utilized tool in biologically-relevant studies. Of these, high lateral resolution methodologies using the NanoSIMS 50/50L have been especially powerful within many biological fields over the past decade. Here, we provide a review of this technology, sample preparation and analysis considerations, examples of recent biological studies, data analysis, and current outlooks. Specifically, we offer an overview of SIMS and development of the NanoSIMS. We describe the major experimental factors that should be considered prior to NanoSIMS analysis and then provide information on best practices for data analysis and image generation, which includesmore » an in-depth discussion of appropriate colormaps. Additionally, we provide an open-source method for data representation that allows simultaneous visualization of secondary electron and ion information within a single image. Lastly, we present a perspective on the future of this technology and where we think it will have the greatest impact in near future.« less
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NanoSIMS for biological applications: Current practices and analyses
DOE Office of Scientific and Technical Information (OSTI.GOV)
Nunez, Jamie R.; Renslow, Ryan S.; Cliff, III, John B.
Secondary ion mass spectrometry (SIMS) has become an increasingly utilized tool in biologically-relevant studies. Of these, high lateral resolution methodologies using the NanoSIMS 50/50L have been especially powerful within many biological fields over the past decade. Here, we provide a review of this technology, sample preparation and analysis considerations, examples of recent biological studies, data analysis, and current outlooks. Specifically, we offer an overview of SIMS and development of the NanoSIMS. We describe the major experimental factors that should be considered prior to NanoSIMS analysis and then provide information on best practices for data analysis and image generation, which includesmore » an in-depth discussion of appropriate colormaps. Additionally, we provide an open-source method for data representation that allows simultaneous visualization of secondary electron and ion information within a single image. Lastly, we present a perspective on the future of this technology and where we think it will have the greatest impact in near future.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Barran, Perdita; Baker, Erin
The great complexity of biological systems and their environment poses similarly vast challenges for accurate analytical evaluations of their identity, structure and quantity. Post genomic science, has predicted much regarding the static populations of biological systems, but a further challenge for analysis is to test the accuracy of these predictions, as well as provide a better representation of the transient nature of the molecules of life. Accurate measurements of biological systems have wide applications for biological, forensic, biotechnological and healthcare fields. Therefore, the holy grail is to find a technique which can identify and quantify biological molecules with high throughput,more » sensitivity and robustness, as well evaluate molecular structure(s) in order to understand how the specific molecules interact and function. While wrapping all of these characteristics into one platform may sound difficult, ion mobility spectrometry (IMS) is addressing all of these challenges. Over the last decade, the number of analytical studies utilizing IMS for the evaluation of complex biological and environmental samples has greatly increased. In most cases IMS is coupled with mass spectrometry (IM-MS), but even alone IMS provides the unique capability of rapidly assessing a molecule’s structure, which can be extremely difficult with other techniques. The robustness of the IMS measurement is bourne out by its widespread use in security, environmental and military applications. The multidimensional IM-MS measurements however have been proven to be ever more powerful, as applied to complex mixtures as they enable the evaluation of both the structure and mass of every molecular component in a sample during a single measurement, without the need for continual reference calibration.« less
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Stolee, Jessica A; Vertes, Akos
2013-04-02
Ambient ionization methods for mass spectrometry have enabled the in situ and in vivo analysis of biological tissues and cells. When an etched optical fiber is used to deliver laser energy to a sample in laser ablation electrospray ionization (LAESI) mass spectrometry, the analysis of large single cells becomes possible. However, because in this arrangement the ablation plume expands in three dimensions, only a small portion of it is ionized by the electrospray. Here we show that sample ablation within a capillary helps to confine the radial expansion of the plume. Plume collimation, due to the altered expansion dynamics, leads to greater interaction with the electrospray plume resulting in increased ionization efficiency, reduced limit of detection (by a factor of ~13, reaching 600 amol for verapamil), and extended dynamic range (6 orders of magnitude) compared to conventional LAESI. This enhanced sensitivity enables the analysis of a range of metabolites from small cell populations and single cells in the ambient environment. This technique has the potential to be integrated with flow cytometry for high-throughput metabolite analysis of sorted cells.
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Long, E; Ilie, M; Lassalle, S; Butori, C; Poissonnet, G; Washetine, K; Mouroux, J; Lespinet, V; Lacour, J P; Taly, V; Laurent-Puig, P; Bahadoran, P; Hofman, V; Hofman, P
2015-12-01
Knowledge of the BRAFV600E status is mandatory in metastatic melanoma patients (MMP). Molecular biology is currently the gold standard method for status assessment. We assessed and compared the specificity, sensibility, cost-effectiveness and turnaround time (TAT) of immunohistochemistry (IHC) and molecular biology for detection of the BRAFV600E mutation in 188 MMP. IHC, with the VE1 antibody, and pyrosequencing analysis were performed with formalin fixed paraffin embedded tumour samples. The BRAFV600E mutation was detected by pyrosequencing in 91/188 (48%) patients. IHC was strongly positive (3+) in all of these 91 cases. IHC was strongly positive in 9/188 (5%) cases in which the molecular testing failed due to non-amplifiable DNA. Weak or moderate staining was noted in 10/188 (5%) cases in which the molecular biology identified BRAF wild-type tumours. The ratio of the global cost for IHC/molecular biology testing was 1 : 2.2. The average TAT was 48 h vs. 96 h, for IHC vs. molecular biology testing, respectively. This study showed that VE1 IHC should be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. This methodology needs to be set up in pathology laboratories in accordance with quality control/quality assurance accreditation procedures. Under these strict conditions the question is to know if BRAFV600E-IHC can serve not only as a prescreening tool, but also as a stand-alone test (at least in cases displaying an unequivocally staining pattern) as well as an alternative predictive test for samples for which the molecular biology failed. © 2015 European Academy of Dermatology and Venereology.
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Integrated Experimental Platforms to Study Blast Injuries: a Bottom-Up Approach
NASA Astrophysics Data System (ADS)
Bo, Chiara
2013-06-01
Developing a cellular and molecular understanding of the nature of traumatic and post-traumatic effects of blast events on live biological samples is critical for improving clinical outcomes.1 To investigate the consequences of pressure waves upon cellular structures and the underlying physiological and biochemical changes, we are using an integrated approach to study the material and biological properties of cells, tissues and organs when subjected to extreme conditions. In particular we have developed a confined Split Hopkinson Pressure Bar (SHPB) system, which allows us to subject cells in suspension or in a monolayer to compression waves of the order of few MPa and duration of hundreds of microseconds.2 The chamber design also enables recovery of the biological samples for cellular and molecular analysis. Specifically, cell survivability, viability, proliferation and morphological changes are investigated post compression for different cell populations. The SHPB platform, coupled with Quasi-Static experiments, is also used to determine stress-strain curves of soft biological tissues under compression at low, medium and high strain rates. Samples are also examined using histological techniques to study macro- and microscopical changes induced by compression waves. Finally, a shock tube has been developed to replicate primary blast damage on organs (i.e. mice lungs) and cell monolayers by generating single or multiple air blast of the order of kPa and few milliseconds duration. This platform allows us to visualize post-traumatic morphological changes at the cellular level as a function of the stimulus pressure and duration as well as biomarker signatures of blast injuries. Adapting and integrating a variety of approaches with different experimental platforms allows us to sample a vast pressure-time space in terms of biological and structural damage that mimic blast injuries and also to determine which physical parameters (peak pressure, stimulus duration, impulse) are contributing to the injury process. Moreover, understanding biological damage following blast events is crucial to developing novel clinical approaches to detect and treat traumatic injury pathologies. This work is supported by he Atomic Weapons Establishment, UK and The Royal British Legion Centre for Blast Injury Studies at Imperial College London, UK
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High-speed single-pixel digital holography
NASA Astrophysics Data System (ADS)
González, Humberto; Martínez-León, Lluís.; Soldevila, Fernando; Araiza-Esquivel, Ma.; Tajahuerce, Enrique; Lancis, Jesús
2017-06-01
The complete phase and amplitude information of biological specimens can be easily determined by phase-shifting digital holography. Spatial light modulators (SLMs) based on liquid crystal technology, with a frame-rate around 60 Hz, have been employed in digital holography. In contrast, digital micro-mirror devices (DMDs) can reach frame rates up to 22 kHz. A method proposed by Lee to design computer generated holograms (CGHs) permits the use of such binary amplitude modulators as phase-modulation devices. Single-pixel imaging techniques record images by sampling the object with a sequence of micro-structured light patterns and using a simple photodetector. Our group has reported some approaches combining single-pixel imaging and phase-shifting digital holography. In this communication, we review these techniques and present the possibility of a high-speed single-pixel phase-shifting digital holography system with phase-encoded illumination. This system is based on a Mach-Zehnder interferometer, with a DMD acting as the modulator for projecting the sampling patterns on the object and also being used for phase-shifting. The proposed sampling functions are phaseencoded Hadamard patterns generated through a Lee hologram approach. The method allows the recording of the complex amplitude distribution of an object at high speed on account of the high frame rates of the DMD. Reconstruction may take just a few seconds. Besides, the optical setup is envisaged as a true adaptive system, which is able to measure the aberration induced by the optical system in the absence of a sample object, and then to compensate the wavefront in the phasemodulation stage.
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Neale, Peta A; Altenburger, Rolf; Aït-Aïssa, Selim; Brion, François; Busch, Wibke; de Aragão Umbuzeiro, Gisela; Denison, Michael S; Du Pasquier, David; Hilscherová, Klára; Hollert, Henner; Morales, Daniel A; Novák, Jiří; Schlichting, Rita; Seiler, Thomas-Benjamin; Serra, Helene; Shao, Ying; Tindall, Andrew J; Tollefsen, Knut Erik; Williams, Timothy D; Escher, Beate I
2017-10-15
Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Scanning transmission electron microscopy through-focal tilt-series on biological specimens.
Trepout, Sylvain; Messaoudi, Cédric; Perrot, Sylvie; Bastin, Philippe; Marco, Sergio
2015-10-01
Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Ucar, Fatma; Erden, Gonul; Ginis, Zeynep; Ozturk, Gulfer; Sezer, Sevilay; Gurler, Mukaddes; Guneyk, Ahmet
2013-10-01
Available data on biological variation of HbA1c revealed marked heterogeneity. We therefore investigated and estimated the components of biological variation for HbA1c in a group of healthy individuals by applying a recommended and strictly designed study protocol using two different assay methods. Each month, samples were derived on the same day, for three months. Four EDTA whole blood samples were collected from each individual (20 women, 9 men; 20-45 years of age) and stored at -80°C until analysis. HbA1c values were measured by both high performance liquid chromatography (HPLC) (Shimadzu, Prominence, Japan) and boronate affinity chromatography methods (Trinity Biotech, Premier Hb9210, Ireland). All samples were assayed in duplicate in a single batch for each assay method. Estimations were calculated according to the formulas described by Fraser and Harris. The within subject (CV(I))-between subject (CV(G)) biological variations were 1.17% and 5.58%, respectively for HPLC. The calculated CV(I) and CV(G) were 2.15% and 4.03%, respectively for boronate affinity chromatography. Reference change value (RCV) for HPLC and boronate affinity chromatography was 5.4% and 10.4% respectively and individuality index of HbA(1c) was 0.35 and 0.93 respectively. This study for the first time described the components of biological variation for HbA1c in healthy individuals by two different assay methods. Obtained findings showed that the difference between CV(A) values of the methods might considerably affect RCV. These data regarding biological variation of HbA(1c) could be useful for a better evaluation of HbA(1c) test results in clinical interpretation. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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Johnson, Nathan T; Dhroso, Andi; Hughes, Katelyn J; Korkin, Dmitry
2018-06-25
The extent to which the genes are expressed in the cell can be simplistically defined as a function of one or more factors of the environment, lifestyle, and genetics. RNA sequencing (RNA-Seq) is becoming a prevalent approach to quantify gene expression, and is expected to gain better insights to a number of biological and biomedical questions, compared to the DNA microarrays. Most importantly, RNA-Seq allows to quantify expression at the gene and alternative splicing isoform levels. However, leveraging the RNA-Seq data requires development of new data mining and analytics methods. Supervised machine learning methods are commonly used approaches for biological data analysis, and have recently gained attention for their applications to the RNA-Seq data. In this work, we assess the utility of supervised learning methods trained on RNA-Seq data for a diverse range of biological classification tasks. We hypothesize that the isoform-level expression data is more informative for biological classification tasks than the gene-level expression data. Our large-scale assessment is done through utilizing multiple datasets, organisms, lab groups, and RNA-Seq analysis pipelines. Overall, we performed and assessed 61 biological classification problems that leverage three independent RNA-Seq datasets and include over 2,000 samples that come from multiple organisms, lab groups, and RNA-Seq analyses. These 61 problems include predictions of the tissue type, sex, or age of the sample, healthy or cancerous phenotypes and, the pathological tumor stage for the samples from the cancerous tissue. For each classification problem, the performance of three normalization techniques and six machine learning classifiers was explored. We find that for every single classification problem, the isoform-based classifiers outperform or are comparable with gene expression based methods. The top-performing supervised learning techniques reached a near perfect classification accuracy, demonstrating the utility of supervised learning for RNA-Seq based data analysis. Published by Cold Spring Harbor Laboratory Press for the RNA Society.
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Methods to Detect Nitric Oxide and its Metabolites in Biological Samples
Bryan, Nathan S.; Grisham, Matthew B.
2007-01-01
Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. NO is involved in many physiological processes including regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification is critical to understanding health and disease. Due to the extremely short physiological half life of this gaseous free radical, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of NO metabolites in biological samples provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The methods described in this review is not an exhaustive or comprehensive discussion of all methods available for the detection of NO but rather a description of the most commonly used and practical methods which allow accurate and sensitive quantification of NO products/metabolites in multiple biological matrices under normal physiological conditions. PMID:17664129
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Yaku, Keisuke; Okabe, Keisuke; Nakagawa, Takashi
2018-06-01
Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism. Copyright © 2018 John Wiley & Sons, Ltd.
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Auerbach, Scott S; Phadke, Dhiral P; Mav, Deepak; Holmgren, Stephanie; Gao, Yuan; Xie, Bin; Shin, Joo Heon; Shah, Ruchir R; Merrick, B Alex; Tice, Raymond R
2015-07-01
Formalin-fixed, paraffin-embedded (FFPE) pathology specimens represent a potentially vast resource for transcriptomic-based biomarker discovery. We present here a comparison of results from a whole transcriptome RNA-Seq analysis of RNA extracted from fresh frozen and FFPE livers. The samples were derived from rats exposed to aflatoxin B1 (AFB1 ) and a corresponding set of control animals. Principal components analysis indicated that samples were separated in the two groups representing presence or absence of chemical exposure, both in fresh frozen and FFPE sample types. Sixty-five percent of the differentially expressed transcripts (AFB1 vs. controls) in fresh frozen samples were also differentially expressed in FFPE samples (overlap significance: P < 0.0001). Genomic signature and gene set analysis of AFB1 differentially expressed transcript lists indicated highly similar results between fresh frozen and FFPE at the level of chemogenomic signatures (i.e., single chemical/dose/duration elicited transcriptomic signatures), mechanistic and pathology signatures, biological processes, canonical pathways and transcription factor networks. Overall, our results suggest that similar hypotheses about the biological mechanism of toxicity would be formulated from fresh frozen and FFPE samples. These results indicate that phenotypically anchored archival specimens represent a potentially informative resource for signature-based biomarker discovery and mechanistic characterization of toxicity. Copyright © 2014 John Wiley & Sons, Ltd.
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NASA Astrophysics Data System (ADS)
Khan, Pintu Kumar; Mahato, Arnab; Kundu, Biswanath; Nandi, Samit K.; Mukherjee, Prasenjit; Datta, Someswar; Sarkar, Soumya; Mukherjee, Jayanta; Nath, Shalini; Balla, Vamsi K.; Mandal, Chitra
2016-09-01
Effects of strontium and lithium ion doping on the biological properties of bioactive glass (BAG) porous scaffolds have been checked in vitro and in vivo. BAG scaffolds were prepared by conventional glass melting route and subsequently, scaffolds were produced by evaporation of fugitive pore formers. After thorough physico-chemical and in vitro cell characterization, scaffolds were used for pre-clinical study. Soft and hard tissue formation in a rabbit femoral defect model after 2 and 4 months, were assessed using different tools. Histological observations showed excellent osseous tissue formation in Sr and Li + Sr scaffolds and moderate bone regeneration in Li scaffolds. Fluorochrome labeling studies showed wide regions of new bone formation in Sr and Li + Sr doped samples as compared to Li doped samples. SEM revealed abundant collagenous network and minimal or no interfacial gap between bone and implant in Sr and Li + Sr doped samples compared to Li doped samples. Micro CT of Li + Sr samples showed highest degree of peripheral cancellous tissue formation on periphery and cortical tissues inside implanted samples and vascularity among four compositions. Our findings suggest that addition of Sr and/or Li alters physico-chemical properties of BAG and promotes early stage in vivo osseointegration and bone remodeling that may offer new insight in bone tissue engineering.
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Khan, Pintu Kumar; Mahato, Arnab; Kundu, Biswanath; Nandi, Samit K.; Mukherjee, Prasenjit; Datta, Someswar; Sarkar, Soumya; Mukherjee, Jayanta; Nath, Shalini; Balla, Vamsi K.; Mandal, Chitra
2016-01-01
Effects of strontium and lithium ion doping on the biological properties of bioactive glass (BAG) porous scaffolds have been checked in vitro and in vivo. BAG scaffolds were prepared by conventional glass melting route and subsequently, scaffolds were produced by evaporation of fugitive pore formers. After thorough physico-chemical and in vitro cell characterization, scaffolds were used for pre-clinical study. Soft and hard tissue formation in a rabbit femoral defect model after 2 and 4 months, were assessed using different tools. Histological observations showed excellent osseous tissue formation in Sr and Li + Sr scaffolds and moderate bone regeneration in Li scaffolds. Fluorochrome labeling studies showed wide regions of new bone formation in Sr and Li + Sr doped samples as compared to Li doped samples. SEM revealed abundant collagenous network and minimal or no interfacial gap between bone and implant in Sr and Li + Sr doped samples compared to Li doped samples. Micro CT of Li + Sr samples showed highest degree of peripheral cancellous tissue formation on periphery and cortical tissues inside implanted samples and vascularity among four compositions. Our findings suggest that addition of Sr and/or Li alters physico-chemical properties of BAG and promotes early stage in vivo osseointegration and bone remodeling that may offer new insight in bone tissue engineering. PMID:27604654
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Cultural interaction and biological distance in postclassic period Mexico.
Ragsdale, Corey S; Edgar, Heather J H
2015-05-01
Economic, political, and cultural relationships connected virtually every population throughout Mexico during Postclassic period (AD 900-1520). Much of what is known about population interaction in prehistoric Mexico is based on archaeological or ethnohistoric data. What is unclear, especially for the Postclassic period, is how these data correlate with biological population structure. We address this by assessing biological (phenotypic) distances among 28 samples based upon a comparison of dental morphology trait frequencies, which serve as a proxy for genetic variation, from 810 individuals. These distances were compared with models representing geographic and cultural relationships among the same groups. Results of Mantel and partial Mantel matrix correlation tests show that shared migration and trade are correlated with biological distances, but geographic distance is not. Trade and political interaction are also correlated with biological distance when combined in a single matrix. These results indicate that trade and political relationships affected population structure among Postclassic Mexican populations. We suggest that trade likely played a major role in shaping patterns of interaction between populations. This study also shows that the biological distance data support the migration histories described in ethnohistoric sources. © 2015 Wiley Periodicals, Inc.
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Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.
Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke
2015-06-11
The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.
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Optical magnetic detection of single-neuron action potentials using quantum defects in diamond
Barry, John F.; Turner, Matthew J.; Schloss, Jennifer M.; Glenn, David R.; Song, Yuyu; Lukin, Mikhail D.; Park, Hongkun; Walsworth, Ronald L.
2016-01-01
Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution—e.g., magnetic resonance imaging methods and magnetoencephalography—or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector. PMID:27911765
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Optical magnetic detection of single-neuron action potentials using quantum defects in diamond.
Barry, John F; Turner, Matthew J; Schloss, Jennifer M; Glenn, David R; Song, Yuyu; Lukin, Mikhail D; Park, Hongkun; Walsworth, Ronald L
2016-12-06
Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution-e.g., magnetic resonance imaging methods and magnetoencephalography-or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector.
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Fontana, F; Rapone, C; Bregola, G; Aversa, R; de Meo, A; Signorini, G; Sergio, M; Ferrarini, A; Lanzellotto, R; Medoro, G; Giorgini, G; Manaresi, N; Berti, A
2017-07-01
Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
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Beck-Jensen, J E; Kollerup, G; Sørensen, H A; Pors Nielsen, S; Sørensen, O H
1997-07-01
Biochemical markers of bone turnover are used to estimate the rate of bone loss in the individual osteoporotic patient. During recent years it has become increasingly clear that the biological variability of biochemical bone markers has to be taken into consideration in the evaluation of their usefulness in the clinical setting. Eleven premenopausal, 8 perimenopausal and 11 postmenopausal healthy women were included. We assessed the analytical and the biological components of variation for a number of resorptive and formative bone markers: u-hydroxyproline, u-pyridinoline, and u-deoxypyridinoline together with u-calcium and u-creatinine, s-total alkaline phosphatases and s-osteocalcin. Blood and urine samples were collected five times with 7-day intervals. Urinary parameters were expressed as outputs and corrected for creatinine in fasting night urines and second void fasting morning urines. The absolute values differed with a tendency towards increasing values in the postmenopausal women, but the biological variations in relation to menopausal status were not different. The biological variability was much higher for the urinary resorptive markers than for the formative markers in the blood. The critical difference expressing the difference needed between two serial results from the same person to be significant at a 5% level was 15% for s-alkaline phosphatases, 18% for s-osteocalcin, and lowest in the second void fasting morning urines with values of 28% and 34% for u-pyridinoline/creatinine and u-deoxypyridinoline/creatinine, and 50% and 112% for u-hydroxyproline/creatinine and u-calcium/creatinine, respectively. The index of individuality, denoting the individual variation divided by the variation between subjects, was in the range from 0.19 for s-alkaline phosphatases to 1.23 for u-hydroxyproline/minute in second void fasting morning urine making the use of conventional reference intervals difficult. Low indices, however, indicate high test performance and offer the possibility of stratification of persons within a range. The number of samples required to determine the true individual mean value +/- 5% for the single person, ranged from 5 for s-total alkaline phosphatases, 6 for s-osteocalcin, 23 for u-deoxypyridinoline/creatinine in the fasting morning urine to over two hundred for u-calcium analytes. It is concluded that, due to high biological variation, a single measurement of biochemical markers of bone turnover is of limited utility in the individual person. We recommend that routine clinical use of biochemical markers should be restricted until further evidence justifies it.
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Single molecule thermodynamics in biological motors.
Taniguchi, Yuichi; Karagiannis, Peter; Nishiyama, Masayoshi; Ishii, Yoshiharu; Yanagida, Toshio
2007-04-01
Biological molecular machines use thermal activation energy to carry out various functions. The process of thermal activation has the stochastic nature of output events that can be described according to the laws of thermodynamics. Recently developed single molecule detection techniques have allowed each distinct enzymatic event of single biological machines to be characterized providing clues to the underlying thermodynamics. In this study, the thermodynamic properties in the stepping movement of a biological molecular motor have been examined. A single molecule detection technique was used to measure the stepping movements at various loads and temperatures and a range of thermodynamic parameters associated with the production of each forward and backward step including free energy, enthalpy, entropy and characteristic distance were obtained. The results show that an asymmetry in entropy is a primary factor that controls the direction in which the motor will step. The investigation on single molecule thermodynamics has the potential to reveal dynamic properties underlying the mechanisms of how biological molecular machines work.
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Multiplex Immunoassay Profiling of Serum in Psychiatric Disorders.
Stephen, Laurie; Schwarz, Emanuel; Guest, Paul C
2017-01-01
Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labour than in single immunoassays. This chapter details the methods to develop and manufacture a 5-plex immunoassay for the Luminex® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. An example will be given for the analysis of five hormones (glucagon-like peptide 1, growth hormone, insulin, leptin and thyroid-stimulating hormone) in serum samples from schizophrenia patients and controls.
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Preservation of Liquid Biological Samples
NASA Technical Reports Server (NTRS)
Putcha, Lakshmi (Inventor); Nimmagudda, Ramalingeshwara (Inventor)
2004-01-01
The present invention related to the preservation of a liquid biological sample. The biological sample is exposed to a preservative containing at least about 0.15 g of sodium benzoate and at least about 0.025 g of citric acid per 100 ml of sample. The biological sample may be collected in a vessel or an absorbent mass. The biological sample may also be exposed to a substrate and/or a vehicle.
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Piegorsch, Walter W.; Lussier, Yves A.
2015-01-01
Motivation: The conventional approach to personalized medicine relies on molecular data analytics across multiple patients. The path to precision medicine lies with molecular data analytics that can discover interpretable single-subject signals (N-of-1). We developed a global framework, N-of-1-pathways, for a mechanistic-anchored approach to single-subject gene expression data analysis. We previously employed a metric that could prioritize the statistical significance of a deregulated pathway in single subjects, however, it lacked in quantitative interpretability (e.g. the equivalent to a gene expression fold-change). Results: In this study, we extend our previous approach with the application of statistical Mahalanobis distance (MD) to quantify personal pathway-level deregulation. We demonstrate that this approach, N-of-1-pathways Paired Samples MD (N-OF-1-PATHWAYS-MD), detects deregulated pathways (empirical simulations), while not inflating false-positive rate using a study with biological replicates. Finally, we establish that N-OF-1-PATHWAYS-MD scores are, biologically significant, clinically relevant and are predictive of breast cancer survival (P < 0.05, n = 80 invasive carcinoma; TCGA RNA-sequences). Conclusion: N-of-1-pathways MD provides a practical approach towards precision medicine. The method generates the magnitude and the biological significance of personal deregulated pathways results derived solely from the patient’s transcriptome. These pathways offer the opportunities for deriving clinically actionable decisions that have the potential to complement the clinical interpretability of personal polymorphisms obtained from DNA acquired or inherited polymorphisms and mutations. In addition, it offers an opportunity for applicability to diseases in which DNA changes may not be relevant, and thus expand the ‘interpretable ‘omics’ of single subjects (e.g. personalome). Availability and implementation: http://www.lussierlab.net/publications/N-of-1-pathways. Contact: yves@email.arizona.edu or piegorsch@math.arizona.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26072495
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A robust method for RNA extraction and purification from a single adult mouse tendon.
Grinstein, Mor; Dingwall, Heather L; Shah, Rishita R; Capellini, Terence D; Galloway, Jenna L
2018-01-01
Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts. Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed. After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons. Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.
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Lee, Jung-Rok; Appelmann, Iris; Miething, Cornelius; Shultz, Tyler O.; Ruderman, Daniel; Kim, Dokyoon; Mallick, Parag; Lowe, Scott W.; Wang, Shan X.
2018-01-01
Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays. Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice. Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment. Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object. PMID:29507628
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Lee, Jung-Rok; Appelmann, Iris; Miething, Cornelius; Shultz, Tyler O; Ruderman, Daniel; Kim, Dokyoon; Mallick, Parag; Lowe, Scott W; Wang, Shan X
2018-01-01
Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays. Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice. Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment. Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object.
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DMRfinder: efficiently identifying differentially methylated regions from MethylC-seq data.
Gaspar, John M; Hart, Ronald P
2017-11-29
DNA methylation is an epigenetic modification that is studied at a single-base resolution with bisulfite treatment followed by high-throughput sequencing. After alignment of the sequence reads to a reference genome, methylation counts are analyzed to determine genomic regions that are differentially methylated between two or more biological conditions. Even though a variety of software packages is available for different aspects of the bioinformatics analysis, they often produce results that are biased or require excessive computational requirements. DMRfinder is a novel computational pipeline that identifies differentially methylated regions efficiently. Following alignment, DMRfinder extracts methylation counts and performs a modified single-linkage clustering of methylation sites into genomic regions. It then compares methylation levels using beta-binomial hierarchical modeling and Wald tests. Among its innovative attributes are the analyses of novel methylation sites and methylation linkage, as well as the simultaneous statistical analysis of multiple sample groups. To demonstrate its efficiency, DMRfinder is benchmarked against other computational approaches using a large published dataset. Contrasting two replicates of the same sample yielded minimal genomic regions with DMRfinder, whereas two alternative software packages reported a substantial number of false positives. Further analyses of biological samples revealed fundamental differences between DMRfinder and another software package, despite the fact that they utilize the same underlying statistical basis. For each step, DMRfinder completed the analysis in a fraction of the time required by other software. Among the computational approaches for identifying differentially methylated regions from high-throughput bisulfite sequencing datasets, DMRfinder is the first that integrates all the post-alignment steps in a single package. Compared to other software, DMRfinder is extremely efficient and unbiased in this process. DMRfinder is free and open-source software, available on GitHub ( github.com/jsh58/DMRfinder ); it is written in Python and R, and is supported on Linux.
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Bacteriophages as Biological Control Agents of Enteric Bacteria Contaminating Edible Oysters.
Le, Tuan Son; Southgate, Paul C; O'Connor, Wayne; Poole, Sue; Kurtbӧke, D Ipek
2018-05-01
Bacterial contamination on seafood resulting from unhygienic food-handling practices causes foodborne diseases and significant revenue losses. Moreover, control measures are complicated by a high prevalence of antibiotic-resistant bacteria. Alternative measures such as the phage therapy, therefore, is considered as an environmental and consumer-friendly biological control strategy for controlling such bacterial contamination. In this study, we determined the effectiveness of a bacteriophage cocktail in controlling E. coli strains [JM 109, ATCC 13706 and the, extended spectrum beta-lactamase resistant strain (ATCC BAA 196)] and S. enterica subsp. enterica (ATCC 13311) as single and combined contaminants of the edible oysters. Five different E. coli-specific phages (belonging to the Siphoviridae family) and a Salmonella phage (belonging to the Tectiviridae family) were successfully isolated from sewage water samples taken from a local sewage treatment plan in the Sunshine Coast region of Australia. Phage treatments applied to the pathogens when they were presented on the oysters as either single or combined hosts, resulted in significant decrease of the number of these bacteria on edible oysters. Results obtained indicated that bacteriophages could have beneficial applications in oyster-processing plants in controlling pathogenic bacterial infestations. This study thus contributes towards ongoing international efforts into the effective use of bacteriophages for biological control purposes.
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NASA Astrophysics Data System (ADS)
Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu
2016-08-01
Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.
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Super-resolution from single photon emission: toward biological application
NASA Astrophysics Data System (ADS)
Moreva, E.; Traina, P.; Forneris, J.; Ditalia Tchernij, S.; Guarina, L.; Franchino, C.; Picollo, F.; Ruo Berchera, I.; Brida, G.; Degiovanni, I. P.; Carabelli, V.; Olivero, P.; Genovese, M.
2017-08-01
Properties of quantum light represent a tool for overcoming limits of classical optics. Several experiments have demonstrated this advantage ranging from quantum enhanced imaging to quantum illumination. In this work, experimental demonstration of quantum-enhanced resolution in confocal fluorescence microscopy will be presented. This is achieved by exploiting the non-classical photon statistics of fluorescence emission of single nitrogen-vacancy (NV) color centers in diamond. By developing a general model of super-resolution based on the direct sampling of the kth-order autocorrelation function of the photoluminescence signal, we show the possibility to resolve, in principle, arbitrarily close emitting centers. Finally, possible applications of NV-based fluorescent nanodiamonds in biosensing and future developments will be presented.
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Review of Detection of Brucella sp. by Polymerase Chain Reaction
Yu, Wei Ling; Nielsen, Klaus
2010-01-01
Here we present a review of most of the currently used polymerase chain reaction (PCR)-based methods for identification of Brucella bacteria in biological samples. We focused in particular on methods using single-pair primers, multiplex primers, real-time PCRs, PCRs for marine Brucella, and PCRs for molecular biotyping. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require minimum biological containment and can provide results in a very short time. In addition, genetic fingerprinting of isolates aid in epidemiological studies of the disease and its control. PCR-based methods are more useful and practical than conventional methods used to identify Brucella spp., and new methods for Brucella spp identification and typing are still being developed. However, the sensitivity, specificity, and issues of quality control and quality assurance using these methods must be fully validated on clinical samples before PCR can be used in routine laboratory testing for brucellosis. PMID:20718083
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Pan, Feng; Liu, Shuo; Wang, Zhe; Shang, Peng; Xiao, Wen
2012-05-07
The long-term and real-time monitoring the cell division and changes of osteoblasts under simulated zero gravity condition were succeed by combing a digital holographic microscopy (DHM) with a superconducting magnet (SM). The SM could generate different magnetic force fields in a cylindrical cavity, where the gravitational force of biological samples could be canceled at a special gravity position by a high magnetic force. Therefore the specimens were levitated and in a simulated zero gravity environment. The DHM was modified to fit with SM by using single mode optical fibers and a vertically-configured jig designed to hold specimens and integrate optical device in the magnet's bore. The results presented the first-phase images of living cells undergoing dynamic divisions and changes under simulated zero gravity environment for a period of 10 hours. The experiments demonstrated that the SM-compatible DHM setup could provide a highly efficient and versatile method for research on the effects of microgravity on biological samples.
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STBase: one million species trees for comparative biology.
McMahon, Michelle M; Deepak, Akshay; Fernández-Baca, David; Boss, Darren; Sanderson, Michael J
2015-01-01
Comprehensively sampled phylogenetic trees provide the most compelling foundations for strong inferences in comparative evolutionary biology. Mismatches are common, however, between the taxa for which comparative data are available and the taxa sampled by published phylogenetic analyses. Moreover, many published phylogenies are gene trees, which cannot always be adapted immediately for species level comparisons because of discordance, gene duplication, and other confounding biological processes. A new database, STBase, lets comparative biologists quickly retrieve species level phylogenetic hypotheses in response to a query list of species names. The database consists of 1 million single- and multi-locus data sets, each with a confidence set of 1000 putative species trees, computed from GenBank sequence data for 413,000 eukaryotic taxa. Two bodies of theoretical work are leveraged to aid in the assembly of multi-locus concatenated data sets for species tree construction. First, multiply labeled gene trees are pruned to conflict-free singly-labeled species-level trees that can be combined between loci. Second, impacts of missing data in multi-locus data sets are ameliorated by assembling only decisive data sets. Data sets overlapping with the user's query are ranked using a scheme that depends on user-provided weights for tree quality and for taxonomic overlap of the tree with the query. Retrieval times are independent of the size of the database, typically a few seconds. Tree quality is assessed by a real-time evaluation of bootstrap support on just the overlapping subtree. Associated sequence alignments, tree files and metadata can be downloaded for subsequent analysis. STBase provides a tool for comparative biologists interested in exploiting the most relevant sequence data available for the taxa of interest. It may also serve as a prototype for future species tree oriented databases and as a resource for assembly of larger species phylogenies from precomputed trees.
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Kuball, Jürgen; Wen, Shu Fen; Leissner, Joachim; Atkins, Derek; Meinhardt, Patricia; Quijano, Erlinda; Engler, Heidrun; Hutchins, Beth; Maneval, Daniel C; Grace, Michael J; Fritz, Mary Ann; Störkel, Stefan; Thüroff, Joachim W; Huber, Christoph; Schuler, Martin
2002-02-15
To study safety, feasibility, and biologic activity of adenovirus-mediated p53 gene transfer in patients with bladder cancer. Twelve patients with histologically confirmed bladder cancer scheduled for cystectomy were treated on day 1 with a single intratumoral injection of SCH 58500 (rAd/p53) at cystoscopy at one dose level (7.5 x 10(11) particles) or a single intravesical instillation of SCH 58500 with a transduction-enhancing agent (Big CHAP) at three dose levels (7.5 x 10(11) to 7.5 x 10(13) particles). Cystectomies were performed in 11 patients on day 3, and transgene expression, vector distribution, and biologic markers of transgene activity were assessed by molecular and immunohistochemical methods in tumors and normal bladder samples. Specific transgene expression was detected in tissues from seven of eight assessable patients treated with intravesical instillation of SCH 58500 but in none of three assessable patients treated with intratumoral injection of SCH 58500. Induction of RNA and protein expression of the p53 target gene p21/WAF1 was demonstrated in samples from patients treated with SCH 58500 instillation at higher dose levels. Distribution studies after intravesical instillation of SCH 58500 revealed both high transduction efficacy and vector penetration throughout the whole urothelium and into submucosal tumor cells. No dose-limiting toxicity was observed, and side effects were local and of transient nature. Intravesical instillation of SCH 58500 combined with a transduction-enhancing agent is safe, feasible, and biologically active in patients with bladder cancer. Studies to evaluate the clinical efficacy of this treatment in patients with localized high-risk bladder cancer are warranted.
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One Bacterial Cell, One Complete Genome
DOE Office of Scientific and Technical Information (OSTI.GOV)
Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos
2010-04-26
While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated frommore » the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.« less
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Localization of single biological molecules out of the focal plane
NASA Astrophysics Data System (ADS)
Gardini, L.; Capitanio, M.; Pavone, F. S.
2014-03-01
Since the behaviour of proteins and biological molecules is tightly related to the cell's environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.
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The collection of MicroED data for macromolecular crystallography.
Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir
2016-05-01
The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.
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4D x-ray phase contrast tomography for repeatable motion of biological samples
NASA Astrophysics Data System (ADS)
Hoshino, Masato; Uesugi, Kentaro; Yagi, Naoto
2016-09-01
X-ray phase contrast tomography based on a grating interferometer was applied to fast and dynamic measurements of biological samples. To achieve this, the scanning procedure in the tomographic scan was improved. A triangle-shaped voltage signal from a waveform generator to a Piezo stage was used for the fast phase stepping in the grating interferometer. In addition, an optical fiber coupled x-ray scientific CMOS camera was used to achieve fast and highly efficient image acquisitions. These optimizations made it possible to perform an x-ray phase contrast tomographic measurement within an 8 min scan with density resolution of 2.4 mg/cm3. A maximum volume size of 13 × 13 × 6 mm3 was obtained with a single tomographic measurement with a voxel size of 6.5 μm. The scanning procedure using the triangle wave was applied to four-dimensional measurements in which highly sensitive three-dimensional x-ray imaging and a time-resolved dynamic measurement of biological samples were combined. A fresh tendon in the tail of a rat was measured under a uniaxial stretching and releasing condition. To maintain the freshness of the sample during four-dimensional phase contrast tomography, the temperature of the bathing liquid of the sample was kept below 10° using a simple cooling system. The time-resolved deformation of the tendon and each fascicle was measured with a temporal resolution of 5.7 Hz. Evaluations of cross-sectional area size, length of the axis, and mass density in the fascicle during a stretching process provided a basis for quantitative analysis of the deformation of tendon fascicle.
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4D x-ray phase contrast tomography for repeatable motion of biological samples.
Hoshino, Masato; Uesugi, Kentaro; Yagi, Naoto
2016-09-01
X-ray phase contrast tomography based on a grating interferometer was applied to fast and dynamic measurements of biological samples. To achieve this, the scanning procedure in the tomographic scan was improved. A triangle-shaped voltage signal from a waveform generator to a Piezo stage was used for the fast phase stepping in the grating interferometer. In addition, an optical fiber coupled x-ray scientific CMOS camera was used to achieve fast and highly efficient image acquisitions. These optimizations made it possible to perform an x-ray phase contrast tomographic measurement within an 8 min scan with density resolution of 2.4 mg/cm 3 . A maximum volume size of 13 × 13 × 6 mm 3 was obtained with a single tomographic measurement with a voxel size of 6.5 μm. The scanning procedure using the triangle wave was applied to four-dimensional measurements in which highly sensitive three-dimensional x-ray imaging and a time-resolved dynamic measurement of biological samples were combined. A fresh tendon in the tail of a rat was measured under a uniaxial stretching and releasing condition. To maintain the freshness of the sample during four-dimensional phase contrast tomography, the temperature of the bathing liquid of the sample was kept below 10° using a simple cooling system. The time-resolved deformation of the tendon and each fascicle was measured with a temporal resolution of 5.7 Hz. Evaluations of cross-sectional area size, length of the axis, and mass density in the fascicle during a stretching process provided a basis for quantitative analysis of the deformation of tendon fascicle.
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High-performance time-resolved fluorescence by direct waveform recording.
Muretta, Joseph M; Kyrychenko, Alexander; Ladokhin, Alexey S; Kast, David J; Gillispie, Gregory D; Thomas, David D
2010-10-01
We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 10(5) times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats.
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Chung, Euiheon; Kim, Daekeun; Cui, Yan; Kim, Yang-Hyo; So, Peter T. C.
2007-01-01
The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or ∼100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy. PMID:17483188
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Highly sensitive detection using microring resonator and nanopores
NASA Astrophysics Data System (ADS)
Bougot-Robin, K.; Hoste, J. W.; Le Thomas, N.; Bienstman, P.; Edel, J. B.
2016-04-01
One of the most significant challenges facing physical and biological scientists is the accurate detection and identification of single molecules in free-solution environments. The ability to perform such sensitive and selective measurements opens new avenues for a large number of applications in biological, medical and chemical analysis, where small sample volumes and low analyte concentrations are the norm. Access to information at the single or few molecules scale is rendered possible by a fine combination of recent advances in technologies. We propose a novel detection method that combines highly sensitive label-free resonant sensing obtained with high-Q microcavities and position control in nanoscale pores (nanopores). In addition to be label-free and highly sensitive, our technique is immobilization free and does not rely on surface biochemistry to bind probes on a chip. This is a significant advantage, both in term of biology uncertainties and fewer biological preparation steps. Through combination of high-Q photonic structures with translocation through nanopore at the end of a pipette, or through a solid-state membrane, we believe significant advances can be achieved in the field of biosensing. Silicon microrings are highly advantageous in term of sensitivity, multiplexing, and microfabrication and are chosen for this study. In term of nanopores, we both consider nanopore at the end of a nanopipette, with the pore being approach from the pipette with nanoprecise mechanical control. Alternatively, solid state nanopores can be fabricated through a membrane, supporting the ring. Both configuration are discussed in this paper, in term of implementation and sensitivity.
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Kocbach, Anette; Li, Yanjun; Yttri, Karl E; Cassee, Flemming R; Schwarze, Per E; Namork, Ellen
2006-01-01
Background Exposure to ambient particulate matter has been associated with a number of adverse health effects. Particle characteristics such as size, surface area and chemistry seem to influence the negative effects of particles. In this study, combustion particles from vehicle exhaust and wood smoke, currently used in biological experiments, were analysed with respect to microstructure and chemistry. Methods Vehicle exhaust particles were collected in a road tunnel during two seasons, with and without use of studded tires, whereas wood smoke was collected from a stove with single-stage combustion. Additionally, a reference diesel sample (SRM 2975) was analysed. The samples were characterised using transmission electron microscopy techniques (TEM/HRTEM, EELS and SAED). Furthermore, the elemental and organic carbon fractions were quantified using thermal optical transmission analysis and the content of selected PAHs was determined by gas chromatography-mass spectrometry. Results Carbon aggregates, consisting of tens to thousands of spherical primary particles, were the only combustion particles identified in all samples using TEM. The tunnel samples also contained mineral particles originating from road abrasion. The geometric diameters of primary carbon particles from vehicle exhaust were found to be significantly smaller (24 ± 6 nm) than for wood smoke (31 ± 7 nm). Furthermore, HRTEM showed that primary particles from both sources exhibited a turbostratic microstructure, consisting of concentric carbon layers surrounding several nuclei in vehicle exhaust or a single nucleus in wood smoke. However, no differences were detected in the graphitic character of primary particles from the two sources using SAED and EELS. The total PAH content was higher for combustion particles from wood smoke as compared to vehicle exhaust, whereas no source difference was found for the ratio of organic to total carbon. Conclusion Combustion particles from vehicle exhaust and residential wood smoke differ in primary particle diameter, microstructure, and PAH content. Furthermore, the analysed samples seem suitable for assessing the influence of physicochemical characteristics of particles on biological responses. PMID:16390554
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NASA Astrophysics Data System (ADS)
Di, Jianglei; Zhao, Jianlin; Sun, Weiwei; Jiang, Hongzhen; Yan, Xiaobo
2009-10-01
Digital holographic microscopy allows the numerical reconstruction of the complex wavefront of samples, especially biological samples such as living cells. In digital holographic microscopy, a microscope objective is introduced to improve the transverse resolution of the sample; however a phase aberration in the object wavefront is also brought along, which will affect the phase distribution of the reconstructed image. We propose here a numerical method to compensate for the phase aberration of thin transparent objects with a single hologram. The least squares surface fitting with points number less than the matrix of the original hologram is performed on the unwrapped phase distribution to remove the unwanted wavefront curvature. The proposed method is demonstrated with the samples of the cicada wings and epidermal cells of garlic, and the experimental results are consistent with that of the double exposure method.
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Solid-phase microextraction technology for in vitro and in vivo metabolite analysis
Zhang, Qihui; Zhou, Liandi; Chen, Hua; Wang, Chong-Zhi; Xia, Zhining; Yuan, Chun-Su
2016-01-01
Analysis of endogenous metabolites in biological samples may lead to the identification of biomarkers in metabolomics studies. To achieve accurate sample analysis, a combined method of continuous quick sampling and extraction is required for online compound detection. Solid-phase microextraction (SPME) integrates sampling, extraction and concentration into a single solvent-free step for chemical analysis. SPME has a number of advantages, including simplicity, high sensitivity and a relatively non-invasive nature. In this article, we reviewed SPME technology in in vitro and in vivo analyses of metabolites after the ingestion of herbal medicines, foods and pharmaceutical agents. The metabolites of microorganisms in dietary supplements and in the gastrointestinal tract will also be examined. As a promising technology in biomedical and pharmaceutical research, SPME and its future applications will depend on advances in analytical technologies and material science. PMID:27695152
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Multiplatform sampling (ship, aircraft, and satellite) of a Gulf Stream warm core ring
NASA Technical Reports Server (NTRS)
Smith, Raymond C.; Brown, Otis B.; Hoge, Frank E.; Baker, Karen S.; Evans, Robert H.
1987-01-01
The purpose of this paper is to demonstrate the ability to meet the need to measure distributions of physical and biological properties of the ocean over large areas synoptically and over long time periods by means of remote sensing utilizing contemporaneous buoy, ship, aircraft, and satellite (i.e., multiplatform) sampling strategies. A mapping of sea surface temperature and chlorophyll fields in a Gulf Stream warm core ring using the multiplatform approach is described. Sampling capabilities of each sensing system are discussed as background for the data collected by means of these three dissimilar methods. Commensurate space/time sample sets from each sensing system are compared, and their relative accuracies in space and time are determined. The three-dimensional composite maps derived from the data set provide a synoptic perspective unobtainable from single platforms alone.
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Optical-fiber-based Mueller optical coherence tomography.
Jiao, Shuliang; Yu, Wurong; Stoica, George; Wang, Lihong V
2003-07-15
An optical-fiber-based multichannel polarization-sensitive Mueller optical coherence tomography (OCT) system was built to acquire the Jones or Mueller matrix of a scattering medium, such as biological tissue. For the first time to our knowledge, fiber-based polarization-sensitive OCT was dynamically calibrated to eliminate the polarization distortion caused by the single-mode optical fiber in the sample arm, thereby overcoming a key technical impediment to the application of optical fibers in this technology. The round-trip Jones matrix of the sampling fiber was acquired from the reflecting surface of the sample for each depth scan (A scan) with our OCT system. A new rigorous algorithm was then used to retrieve the calibrated polarization properties of the sample. This algorithm was validated with experimental data. The skin of a rat was imaged with this fiber-based system.
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Analyzing cell fate control by cytokines through continuous single cell biochemistry.
Rieger, Michael A; Schroeder, Timm
2009-10-01
Cytokines are important regulators of cell fates with high clinical and commercial relevance. However, despite decades of intense academic and industrial research, it proved surprisingly difficult to describe the biological functions of cytokines in a precise and comprehensive manner. The exact analysis of cytokine biology is complicated by the fact that individual cytokines control many different cell fates and activate a multitude of intracellular signaling pathways. Moreover, although activating different molecular programs, different cytokines can be redundant in their biological effects. In addition, cytokines with different biological effects can activate overlapping signaling pathways. This prospect article will outline the necessity of continuous single cell biochemistry to unravel the biological functions of molecular cytokine signaling. It focuses on potentials and limitations of recent technical developments in fluorescent time-lapse imaging and single cell tracking allowing constant long-term observation of molecules and behavior of single cells. (c) 2009 Wiley-Liss, Inc.
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Hayashi, Tetsutaro; Ozaki, Haruka; Sasagawa, Yohei; Umeda, Mana; Danno, Hiroki; Nikaido, Itoshi
2018-02-12
Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.
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Clime, Liviu; Hoa, Xuyen D; Corneau, Nathalie; Morton, Keith J; Luebbert, Christian; Mounier, Maxence; Brassard, Daniel; Geissler, Matthias; Bidawid, Sabah; Farber, Jeff; Veres, Teodor
2015-02-01
Detecting pathogenic bacteria in food or other biological samples with lab-on-a-chip (LOC) devices requires several sample preparation steps prior to analysis which commonly involves cleaning complex sample matrices of large debris. This often underestimated step is important to prevent these larger particles from clogging devices and to preserve initial concentrations when LOC techniques are used to concentrate or isolate smaller target microorganisms for downstream analysis. In this context, we developed a novel microfluidic system for membrane-free cleaning of biological samples from debris particles by combining hydrodynamic focusing and inertial lateral migration effects. The microfluidic device is fabricated using thermoplastic elastomers being compatible with thermoforming fabrication techniques leading to low-cost single-use devices. Microfluidic chip design and pumping protocols are optimized by investigating diffusive losses numerically with coupled Navier-Stokes and convective-diffusion theoretical models. Stability of inertial lateral migration and separation of debris is assessed through fluorescence microscopy measurements with labelled particles serving as a model system. Efficiency of debris cleaning is experimentally investigated by monitoring microchip outlets with in situ optical turbidity sensors, while retention of targeted pathogens (i.e., Listeria monocytogenes) within the sample stream is assessed through bacterial culture techniques. Optimized pumping protocols can remove up to 50 % of debris from ground beef samples while percentage for preserved microorganisms can account for 95 % in relatively clean samples. However, comparison between inoculated turbid and clean samples (i.e., with and without ground beef debris) indicate some degree of interference between debris inertial lateral migration and hydrodynamic focusing of small microorganisms. Although this interference can lead to significant decrease in chip performance through loss of target bacteria, it remains possible to reach 70 % for sample recovery and more than 50 % for debris removal even in the most turbid samples tested. Due to the relatively simple design, the robustness of the inertial migration effect itself, the high operational flow rates and fabrication methods that leverage low-cost materials, the proposed device can have an impact on a wide range of applications where high-throughput separation of particles and biological species is of interest.
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Generation of light-sheet at the end of multimode fibre (Conference Presentation)
NASA Astrophysics Data System (ADS)
Plöschner, Martin; Kollárová, Véra; Dostál, Zbyněk.; Nylk, Jonathan; Barton-Owen, Thomas; Ferrier, David E. K.; Chmelik, Radim; Dholakia, Kishan; Cizmár, TomáÅ.¡
2017-02-01
Light-sheet fluorescence microscopy is quickly becoming one of the cornerstone imaging techniques in biology as it provides rapid, three-dimensional sectioning of specimens at minimal levels of phototoxicity. It is very appealing to bring this unique combination of imaging properties into an endoscopic setting and be able to perform optical sectioning deep in tissues. Current endoscopic approaches for delivery of light-sheet illumination are based on single-mode optical fibre terminated by cylindrical gradient index lens. Such configuration generates a light-sheet plane that is axially fixed and a mechanical movement of either the sample or the endoscope is required to acquire three-dimensional information about the sample. Furthermore, the axial resolution of this technique is limited to 5um. The delivery of the light-sheet through the multimode fibre provides better axial resolution limited only by its numerical aperture, the light-sheet is scanned holographically without any mechanical movement, and multiple advanced light-sheet imaging modalities, such as Bessel and structured illumination Bessel beam, are intrinsically supported by the system due to the cylindrical symmetry of the fibre. We discuss the holographic techniques for generation of multiple light-sheet types and demonstrate the imaging on a sample of fluorescent beads fixed in agarose gel, as well as on a biological sample of Spirobranchus Lamarcki.
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Pudda, Catherine; Boizot, François; Verplanck, Nicolas; Revol-Cavalier, Frédéric; Berthier, Jean; Thuaire, Aurélie
2018-01-01
Particle separation in microfluidic devices is a common problematic for sample preparation in biology. Deterministic lateral displacement (DLD) is efficiently implemented as a size-based fractionation technique to separate two populations of particles around a specific size. However, real biological samples contain components of many different sizes and a single DLD separation step is not sufficient to purify these complex samples. When connecting several DLD modules in series, pressure balancing at the DLD outlets of each step becomes critical to ensure an optimal separation efficiency. A generic microfluidic platform is presented in this paper to optimize pressure balancing, when DLD separation is connected either to another DLD module or to a different microfluidic function. This is made possible by generating droplets at T-junctions connected to the DLD outlets. Droplets act as pressure controllers, which perform at the same time the encapsulation of DLD sorted particles and the balance of output pressures. The optimized pressures to apply on DLD modules and on T-junctions are determined by a general model that ensures the equilibrium of the entire platform. The proposed separation platform is completely modular and reconfigurable since the same predictive model applies to any cascaded DLD modules of the droplet-based cartridge. PMID:29768490
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Single track effects, Biostack and risk assessment
NASA Technical Reports Server (NTRS)
Curtis, S. B.; Chatterjee, A. (Principal Investigator)
1994-01-01
The scientific career of Prof. Bucker has spanned a very exciting period in the fledgling science of Space Radiation Biology. The capability for placing biological objects in space was developed, and the methods for properly packaging, retrieving and analyzing them were worked out. Meaningful results on the effects of radiation were obtained for the first time. In fact, many of the successful techniques and methodologies for handling biological samples were developed in Prof. Bucker's laboratories, as attested by the extensive Biostack program. He was the first to suggest and successfully carry out experiments in space directly aimed at measuring effects of single tracks of high-energy heavy galactic cosmic rays by specifically identifying whether or not the object had been hit by a heavy particle track. Because the "hit" frequencies of heavy galactic cosmic rays to cell nuclei in the bodies of space travelers will be low, it is expected that any effects to humans on the cellular level will be dominated by single-track cell traversals. This includes the most important generally recognized late effect of space radiation exposure: radiation-induced cancer. This paper addresses the single-track nature of the space radiation environment, and points out the importance of single "hits" in the evaluation of radiation risk for long-term missions occurring outside the earth's magnetic field. A short review is made of biological objects found to show increased effects when "hit" by a single heavy charged-particle in space. A brief discussion is given of the most provocative results from the bacterial spore Bacillus subtilis: experimental evidence that tracks can affect biological systems at much larger distances from the trajectory than previously suspected, and that the resultant inactivation cross section in space calculated for this system is very large. When taken at face value, the implication of these results, when compared to those from experiments performed at ground-based accelerators with beams at low energies in the same LET range, is that high-energy particles can exert their influence a surprising distance from their trajectory and the inactivation cross sections are some 20 times larger than expected. Clearly, beams from high-energy heavy-ion accelerators should be used to confirm these results. For those end points that can also be caused by low-LET beams such as high-energy protons, it is important to measure their action cross sections as well. The ratio of the cross sections for a high-LET beam to that of a low-LET beam is an interesting experimental ratio and, we suggest, of more intrinsic interest than the RBE (Relative Biological Effectiveness). It is a measure of the "biological" importance of one particle type relative to another particle type. This ratio will be introduced and given the name RPPE (Relative Per Particle Effectiveness). Values of RPPE have appeared in the literature and will be discussed. A rather well-known value of this quantity (13,520) has been suggested for the RPPE of high-energy iron ions to high-energy protons. This value was suggested by Letaw et al. Nature 330, 709-710 (1987)] we will call it the Letaw limit. It will be discussed in terms of the importance of the heavy-ion component vs light-ion component of the galactic cosmic rays. It is also pointed out, however, that there may be unique effects from single tracks of heavy ions that do not occur from light-ion tracks. For such effects, the concepts of both RBE and RPPE lose their meaning.
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Imaging and Force Recognition of Single Molecular Behaviors Using Atomic Force Microscopy
Li, Mi; Dang, Dan; Liu, Lianqing; Xi, Ning; Wang, Yuechao
2017-01-01
The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed. PMID:28117741
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NASA Astrophysics Data System (ADS)
Branson, O.; Vetter, L.; Fehrenbacher, J. S.; Spero, H. J.
2016-12-01
The geochemical variability between individual foraminifera within single core intervals records both palaeo-oecanographic conditions and ecology. Within the biological context of foraminiferal species, this population variability may be interpreted to provide unparalleled paleoenvironmental information. For example, coupled trace element and stable isotope analyses of single O. universa offer a powerful tool for reconstructing the δ18O of Laurentide Ice Sheet (LIS) meltwater, by calculating the intercept between temperature-corrected δ18O water and Ba/Ca salinity estimates (Vetter et al., in review). This offers valuable insights into the dynamics of ice sheet melting at the end of the last glacial maximum. Here we apply similar coupled single-shell laser ablation (LA-ICP-MS) and isotope ratio mass spectrometry (IRMS) techniques to explore the δ18O of Laurentide meltwater during H4 and bracketing intervals. The application of these methods to down-core samples requires the development of robust LA-ICP-MS data processing techniques to identify primary signals within Ba contaminated samples, and careful consideration of palaeo Ba/Ca-salinity relationships. Our analyses offer a significant advance in systematic LA-ICP-MS data processing methods, offer constraints on the variability of riverine Ba fluxes, and ultimately provide δ18O estimates of LIS meltwater during H4.
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ERIC Educational Resources Information Center
Zhang, Xu; Shao, Meng; Gao, Lu; Zhao, Yuanyuan; Sun, Zixuan; Zhou, Liping; Yan, Yongmin; Shao, Qixiang; Xu, Wenrong; Qian, Hui
2017-01-01
Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of…
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NASA Astrophysics Data System (ADS)
Schwartz, Jerrod J.; Stavrakis, Stavros; Quake, Stephen R.
2010-02-01
Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room-temperature observations, largely owing to the use of objective lenses with high numerical aperture, brighter fluorophores and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single-molecule level both in vitro and in vivo. However, it is difficult to study systems and phenomena at temperatures above 37 °C, because the index-matching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens, and sustained exposure to high temperatures can cause the lens to fail. Here, we report that TiO2 colloids with diameters of 2 µm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 °C when placed in immediate proximity to an emitting molecule. The optical system is completed by a low-numerical-aperture optic that can have a long working distance and an air interface, which allows the sample to be independently heated. Colloidal lenses were used for parallel imaging of surface-immobilized single fluorophores and for real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 °C. Fluorophores in close proximity to TiO2 also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.
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NASA Astrophysics Data System (ADS)
Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.
2014-08-01
Primary biological aerosol particles (PBAPs) can contribute significantly to the coarse particle burden in many environments. PBAPs can thus influence climate and precipitation systems as cloud nuclei and can spread disease to humans, animals, and plants. Measurement data and techniques for PBAPs in natural environments at high time- and size resolution are, however, sparse, and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in southwestern Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of a waveband integrated bioaerosol sensor (WIBS-4) with a ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behavior, with increased fluorescent bioparticle concentrations at night, when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each was correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multimodal distributions turning into a broad featureless single mode after averaging, and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are relatively insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse-mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAPs observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.
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NASA Astrophysics Data System (ADS)
Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.
2014-02-01
Primary biological aerosol particles (PBAP) can contribute significantly to the coarse particle burden in many environments, may thus influence climate and precipitation systems as cloud nuclei, and can spread disease to humans, animals, and plants. Measurements of PBAP in natural environments taken at high time- and size- resolution are, however, sparse and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in south western Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of the waveband integrated bioaerosol sensor (WIBS-4) with the ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behaviour, with increased fluorescent bioparticle concentrations at night when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each were correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multi-modal distributions turning into a broad featureless single mode after averaging and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAP observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.
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Multi-distance diffuse optical spectroscopy with a single optode via hypotrochoidal scanning.
Applegate, Matthew B; Roblyer, Darren
2018-02-15
Frequency-domain diffuse optical spectroscopy (FD-DOS) is an established technique capable of determining optical properties and chromophore concentrations in biological tissue. Most FD-DOS systems use either manually positioned, handheld probes or complex arrays of source and detector fibers to acquire data from many tissue locations, allowing for the generation of 2D or 3D maps of tissue. Here, we present a new method to rapidly acquire a wide range of source-detector (SD) separations by mechanically scanning a single SD pair. The source and detector fibers are mounted on a scan head that traces a hypotrochoidal pattern over the sample that, when coupled with a high-speed FD-DOS system, enables the rapid collection of dozens of SD separations for depth-resolved imaging. We demonstrate that this system has an average error of 4±2.6% in absorption and 2±1.8% in scattering across all SD separations. Additionally, by linearly translating the device, the size and location of an absorbing inhomogeneity can be determined through the generation of B-scan images in a manner conceptually analogous to ultrasound imaging. This work demonstrates the potential of single optode diffuse optical scanning for depth resolved visualization of heterogeneous biological tissues at near real-time rates.
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The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform
Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott
2013-01-01
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.
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Improved Time-Lapsed Angular Scattering Microscopy of Single Cells
NASA Astrophysics Data System (ADS)
Cannaday, Ashley E.
By measuring angular scattering patterns from biological samples and fitting them with a Mie theory model, one can estimate the organelle size distribution within many cells. Quantitative organelle sizing of ensembles of cells using this method has been well established. Our goal is to develop the methodology to extend this approach to the single cell level, measuring the angular scattering at multiple time points and estimating the non-nuclear organelle size distribution parameters. The diameters of individual organelle-size beads were successfully extracted using scattering measurements with a minimum deflection angle of 20 degrees. However, the accuracy of size estimates can be limited by the angular range detected. In particular, simulations by our group suggest that, for cell organelle populations with a broader size distribution, the accuracy of size prediction improves substantially if the minimum angle of detection angle is 15 degrees or less. The system was therefore modified to collect scattering angles down to 10 degrees. To confirm experimentally that size predictions will become more stable when lower scattering angles are detected, initial validations were performed on individual polystyrene beads ranging in diameter from 1 to 5 microns. We found that the lower minimum angle enabled the width of this delta-function size distribution to be predicted more accurately. Scattering patterns were then acquired and analyzed from single mouse squamous cell carcinoma cells at multiple time points. The scattering patterns exhibit angular dependencies that look unlike those of any single sphere size, but are well-fit by a broad distribution of sizes, as expected. To determine the fluctuation level in the estimated size distribution due to measurement imperfections alone, formaldehyde-fixed cells were measured. Subsequent measurements on live (non-fixed) cells revealed an order of magnitude greater fluctuation in the estimated sizes compared to fixed cells. With our improved and better-understood approach to single cell angular scattering, we are now capable of reliably detecting changes in organelle size predictions due to biological causes above our measurement error of 20 nm, which enables us to apply our system to future studies of the investigation of various single cell biological processes.
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Multi-Omics Factor Analysis-a framework for unsupervised integration of multi-omics data sets.
Argelaguet, Ricard; Velten, Britta; Arnol, Damien; Dietrich, Sascha; Zenz, Thorsten; Marioni, John C; Buettner, Florian; Huber, Wolfgang; Stegle, Oliver
2018-06-20
Multi-omics studies promise the improved characterization of biological processes across molecular layers. However, methods for the unsupervised integration of the resulting heterogeneous data sets are lacking. We present Multi-Omics Factor Analysis (MOFA), a computational method for discovering the principal sources of variation in multi-omics data sets. MOFA infers a set of (hidden) factors that capture biological and technical sources of variability. It disentangles axes of heterogeneity that are shared across multiple modalities and those specific to individual data modalities. The learnt factors enable a variety of downstream analyses, including identification of sample subgroups, data imputation and the detection of outlier samples. We applied MOFA to a cohort of 200 patient samples of chronic lymphocytic leukaemia, profiled for somatic mutations, RNA expression, DNA methylation and ex vivo drug responses. MOFA identified major dimensions of disease heterogeneity, including immunoglobulin heavy-chain variable region status, trisomy of chromosome 12 and previously underappreciated drivers, such as response to oxidative stress. In a second application, we used MOFA to analyse single-cell multi-omics data, identifying coordinated transcriptional and epigenetic changes along cell differentiation. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.
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Kim Tiam, Sandra; Fauvelle, Vincent; Morin, Soizic; Mazzella, Nicolas
2016-01-01
Complexity of contaminants exposure needs to be taking in account for an appropriate evaluation of risks related to mixtures of pesticides released in the ecosystems. Toxicity assessment of such mixtures can be made through a variety of toxicity tests reflecting different level of biological complexity. This paper reviews the recent developments of passive sampling techniques for polar compounds, especially Polar Organic Chemical Integrative Samplers (POCIS) and Chemcatcher® and the principal assessment techniques using microalgae in laboratory experiments. The progresses permitted by the coupled use of such passive samplers and ecotoxicology testing as well as their limitations are presented. Case studies combining passive sampling devices (PSD) extracts and toxicity assessment toward microorganisms at different biological scales from single organisms to communities level are presented. These case studies, respectively, aimed (i) at characterizing the “toxic potential” of waters using dose-response curves, and (ii) at performing microcosm experiments with increased environmental realism in the toxicant exposure in term of cocktail composition and concentration. Finally perspectives and limitations of such approaches for future applications in the area of environmental risk assessment are discussed. PMID:27667986
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Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T
2015-04-03
Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni
2015-01-01
Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we have shown that folded proteins can be internalised inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalisation of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement, and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores might provide a new approach to probe conformational heterogeneity in protein:DNA interactions. PMID:25493908
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Low-energy electron holographic imaging of individual tobacco mosaic virions
DOE Office of Scientific and Technical Information (OSTI.GOV)
Longchamp, Jean-Nicolas, E-mail: longchamp@physik.uzh.ch; Latychevskaia, Tatiana; Escher, Conrad
2015-09-28
Modern structural biology relies on Nuclear Magnetic Resonance (NMR), X-ray crystallography, and cryo-electron microscopy for gaining information on biomolecules at nanometer, sub-nanometer, or atomic resolution. All these methods, however, require averaging over a vast ensemble of entities, and hence knowledge on the conformational landscape of an individual particle is lost. Unfortunately, there are now strong indications that even X-ray free electron lasers will not be able to image individual molecules but will require nanocrystal samples. Here, we show that non-destructive structural biology of single particles has now become possible by means of low-energy electron holography. As an example, individual tobaccomore » mosaic virions deposited on ultraclean freestanding graphene are imaged at 1 nm resolution revealing structural details arising from the helical arrangement of the outer protein shell of the virus. Since low-energy electron holography is a lens-less technique and since electrons with a deBroglie wavelength of approximately 1 Å do not impose radiation damage to biomolecules, the method has the potential for Angstrom resolution imaging of single biomolecules.« less
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Modified rare earth semiconductor oxide as a new nucleotide probe.
Shrestha, S; Mills, C E; Lewington, J; Tsang, S C
2006-12-28
Recent rapid developments in biological analysis, medical diagnosis, pharmaceutical industry, and environmental control fuel the urgent need for recognition of particular DNA sequences from samples. Currently, DNA detection techniques use radiochemical, enzymatic, fluorescent, or electrochemiluminescent methods; however, these techniques require costly labeled DNA and highly skilled and cumbersome procedure, which prohibit any in-situ monitoring. Here, we report that hybridization of surface-immobilized single-stranded oligonucleotide on praseodymium oxide (evaluated as a biosensor surface for the first time) with complimentary strands in solution provokes a significant shift of electrical impedance curve. This shift is attributed to a change in electrical characteristics through modification of surface charge of the underlying modified praseodymium oxide upon hybridization with the complementary oligonucelotide strand. On the other hand, using a noncomplementary single strand in solution does not create an equivalent change in the impedance value. This result clearly suggests that a new and simple electrochemical technique based on the change in electrical properties of the modified praseodymium oxide semiconductor surface upon recognition and transduction of a biological event without using labeled species is revealed.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hanamura, S.; Smith, B.W.; Winefordner, J.D.
1983-11-01
By means of thermal vaporization, inorganic, organic, and metallorganic species are separated and elemental emission in a microwave plasma is detected as a function of vaporization temperature. Solid samples of 250 mg or more are used to avoid problems with sample heterogeneity. The precision of characteristic appearance temperatures is +/-2/sup 0/C. The single electrode atmosphere pressure microwave plasma system is extremely tolerant to the introduction of water, organic solvents, and air. The measurement system contained a repetition wavelength scan device to allow background correction. The plasma temperature was 5500 K. The system was used to measure C, H, N, O,more » and Hg in orchard leaves and in tuna fish. 9 figures, 5 tables.« less
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Coller, Hilary A
2017-09-01
Emerging technologies for the analysis of genome-wide information in single cells have the potential to transform many fields of biology, including our understanding of cell states, the response of cells to external stimuli, mosaicism, and intratumor heterogeneity. At Experimental Biology 2017 in Chicago, Physiological Genomics hosted a symposium in which five leaders in the field of single cell genomics presented their recent research. The speakers discussed emerging methodologies in single cell analysis and critical issues for the analysis of single cell data. Also discussed were applications of single cell genomics to understanding the different types of cells within an organism or tissue and the basis for cell-to-cell variability in response to stimuli. Copyright © 2017 the American Physiological Society.
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A Problem-Sorting Task Detects Changes in Undergraduate Biological Expertise over a Single Semester
Hoskinson, Anne-Marie; Maher, Jessica Middlemis; Bekkering, Cody; Ebert-May, Diane
2017-01-01
Calls for undergraduate biology reform share similar goals: to produce people who can organize, use, connect, and communicate about biological knowledge. Achieving these goals requires students to gain disciplinary expertise. Experts organize, access, and apply disciplinary knowledge differently than novices, and expertise is measurable. By asking introductory biology students to sort biological problems, we investigated whether they changed how they organized and linked biological ideas over one semester of introductory biology. We administered the Biology Card Sorting Task to 751 students enrolled in their first or second introductory biology course focusing on either cellular–molecular or organismal–population topics, under structured or unstructured sorting conditions. Students used a combination of superficial, deep, and yet-uncharacterized ways of organizing and connecting biological knowledge. In some cases, this translated to more expert-like ways of organizing knowledge over a single semester, best predicted by whether students were enrolled in their first or second semester of biology and by the sorting condition completed. In addition to illuminating differences between novices and experts, our results show that card sorting is a robust way of detecting changes in novices’ biological expertise—even in heterogeneous populations of novice biology students over the time span of a single semester. PMID:28408406
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Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.
Stephen, Laurie; Guest, Paul C
2018-01-01
Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.
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Multiplex Immunoassay Profiling.
Stephen, Laurie
2017-01-01
Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.
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High resolution axicon-based endoscopic FD OCT imaging with a large depth range
NASA Astrophysics Data System (ADS)
Lee, Kye-Sung; Hurley, William; Deegan, John; Dean, Scott; Rolland, Jannick P.
2010-02-01
Endoscopic imaging in tubular structures, such as the tracheobronchial tree, could benefit from imaging optics with an extended depth of focus (DOF). This optics could accommodate for varying sizes of tubular structures across patients and along the tree within a single patient. In the paper, we demonstrate an extended DOF without sacrificing resolution showing rotational images in biological tubular samples with 2.5 μm axial resolution, 10 ìm lateral resolution, and > 4 mm depth range using a custom designed probe.
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Polarization sensitive localization based super-resolution microscopy with a birefringent wedge
NASA Astrophysics Data System (ADS)
Sinkó, József; Gajdos, Tamás; Czvik, Elvira; Szabó, Gábor; Erdélyi, Miklós
2017-03-01
A practical method has been presented for polarization sensitive localization based super-resolution microscopy using a birefringent dual wedge. The measurement of the polarization degree at the single molecule level can reveal the chemical and physical properties of the local environment of the fluorescent dye molecule and can hence provide information about the sub-diffraction sized structure of biological samples. Polarization sensitive STORM imaging of the F-Actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.
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Nawrocki, Eric P.; Burge, Sarah W.
2013-01-01
The development of RNA bioinformatic tools began more than 30 y ago with the description of the Nussinov and Zuker dynamic programming algorithms for single sequence RNA secondary structure prediction. Since then, many tools have been developed for various RNA sequence analysis problems such as homology search, multiple sequence alignment, de novo RNA discovery, read-mapping, and many more. In this issue, we have collected a sampling of reviews and original research that demonstrate some of the many ways bioinformatics is integrated with current RNA biology research. PMID:23948768
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Technical aspects and recommendations for single-cell qPCR.
Ståhlberg, Anders; Kubista, Mikael
2018-02-01
Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.
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From the iron boring scraps to superparamagnetic nanoparticles through an aerobic biological route.
Daneshvar, Majid; Hosseini, Mohammad Raouf
2018-06-15
A straightforward, highly efficient, and low-cost biological route was introduced for the synthesis of magnetic nanoparticles. Three urease-positive bacteria namely, Bacillus subtilis, B. pasteurii, and B. licheniformis were used to biosynthesize ammonia and biosurfactants required for the nanoparticle production. Also, the features of the applied biological approach was compared with a chemical co-precipitation method. X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), vibrating-sample magnetometer (VSM), and Fourier transform infrared spectroscopy (FTIR) were applied to characterize the synthesized nanoparticles. Results indicated that the biologically fabricated powders had a single domain structure, and their mean particle size was in the range of 37 to 97 nm. The production capacity of the biological processes was double the chemical method, and the biosynthesized superparamagnetic nanoparticles had higher saturation magnetization up to 132 emu/g. Finally, the removal of Cr(VI) from a synthetic solution was investigated using the four products. The maximum elimination of chromium (over 99%) was achieved by the particles synthesized by B. pasteurii, with the adsorption capacity of 190 mg/g. Copyright © 2018 Elsevier B.V. All rights reserved.
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Multispectral optical tweezers for molecular diagnostics of single biological cells
NASA Astrophysics Data System (ADS)
Butler, Corey; Fardad, Shima; Sincore, Alex; Vangheluwe, Marie; Baudelet, Matthieu; Richardson, Martin
2012-03-01
Optical trapping of single biological cells has become an established technique for controlling and studying fundamental behavior of single cells with their environment without having "many-body" interference. The development of such an instrument for optical diagnostics (including Raman and fluorescence for molecular diagnostics) via laser spectroscopy with either the "trapping" beam or secondary beams is still in progress. This paper shows the development of modular multi-spectral imaging optical tweezers combining Raman and Fluorescence diagnostics of biological cells.
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NASA Astrophysics Data System (ADS)
Bogan, Michael J.; Starodub, Dmitri; Hampton, Christina Y.; Sierra, Raymond G.
2010-10-01
The first of its kind, the Free electron LASer facility in Hamburg, FLASH, produces soft x-ray pulses with unprecedented properties (10 fs, 6.8-47 nm, 1012 photons per pulse, 20 µm diameter). One of the seminal FLASH experiments is single-pulse coherent x-ray diffractive imaging (CXDI). CXDI utilizes the ultrafast and ultrabright pulses to overcome resolution limitations in x-ray microscopy imposed by x-ray-induced damage to the sample by 'diffracting before destroying' the sample on sub-picosecond timescales. For many lensless imaging algorithms used for CXDI it is convenient when the data satisfy an oversampling constraint that requires the sample to be an isolated object, i.e. an individual 'free-standing' portion of disordered matter delivered to the centre of the x-ray focus. By definition, this type of matter is an aerosol. This paper will describe the role of aerosol science methodologies used for the validation of the 'diffract before destroy' hypothesis and the execution of the first single-particle CXDI experiments being developed for biological imaging. FLASH CXDI now enables the highest resolution imaging of single micron-sized or smaller airborne particulate matter to date while preserving the native substrate-free state of the aerosol. Electron microscopy offers higher resolution for single-particle analysis but the aerosol must be captured on a substrate, potentially modifying the particle morphology. Thus, FLASH is poised to contribute significant advancements in our knowledge of aerosol morphology and dynamics. As an example, we simulate CXDI of combustion particle (soot) morphology and introduce the concept of extracting radius of gyration of fractal aggregates from single-pulse x-ray diffraction data. Future upgrades to FLASH will enable higher spatially and temporally resolved single-particle aerosol dynamics studies, filling a critical technological need in aerosol science and nanotechnology. Many of the methodologies described for FLASH will directly translate to use at hard x-ray free electron lasers.
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Leveraging the rice genome sequence for monocot comparative and translational genomics.
Lohithaswa, H C; Feltus, F A; Singh, H P; Bacon, C D; Bailey, C D; Paterson, A H
2007-07-01
Common genome anchor points across many taxa greatly facilitate translational and comparative genomics and will improve our understanding of the Tree of Life. To add to the repertoire of genomic tools applicable to the study of monocotyledonous plants in general, we aligned Allium and Musa ESTs to Oryza BAC sequences and identified candidate Allium-Oryza and Musa-Oryza conserved intron-scanning primers (CISPs). A random sampling of 96 CISP primer pairs, representing loci from 11 of the 12 chromosomes in rice, were tested on seven members of the order Poales and on representatives of the Arecales, Asparagales, and Zingiberales monocot orders. The single-copy amplification success rates of Allium (31.3%), Cynodon (31.4%), Hordeum (30.2%), Musa (37.5%), Oryza (61.5%), Pennisetum (33.3%), Sorghum (47.9%), Zea (33.3%), Triticum (30.2%), and representatives of the palm family (32.3%) suggest that subsets of these primers will provide DNA markers suitable for comparative and translational genomics in orphan crops, as well as for applications in conservation biology, ecology, invasion biology, population biology, systematic biology, and related fields.
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Kalin, Robert M.; Hamilton, John T.G.; Harper, David B.; Miller, Laurence G.; Lamb, Clare; Kennedy, James T.; Downey, Angela; McCauley, Sean; Goldstein, Allen H.
2001-01-01
Gas chromatography/mass spectrometry/isotope ratio mass spectrometry (GC/MS/IRMS) methods for δ13C measurement of the halomethanes CH3Cl, CH3Br, CH3I and methanethiol (CH3SH) during studies of their biological production, biological degradation, and abiotic reactions are presented. Optimisation of gas chromatographic parameters allowed the identification and quantification of CO2, O2, CH3Cl, CH3Br, CH3I and CH3SH from a single sample, and also the concurrent measurement of δ13C for each of the halomethanes and methanethiol. Precision of δ13C measurements for halomethane standards decreased (±0.3, ±0.5 and ±1.3‰) with increasing mass (CH3Cl, CH3Br, CH3I, respectively). Given that carbon isotope effects during biological production, biological degradation and some chemical (abiotic) reactions can be as much as 100‰, stable isotope analysis offers a precise method to study the global sources and sinks of these halogenated compounds that are of considerable importance to our understanding of stratospheric ozone destruction.
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Afornali, Alessandro; de Vecchi, Rodrigo; Stuart, Rodrigo Makowiecky; Dieamant, Gustavo; de Oliveira, Luciana Lima; Brohem, Carla Abdo; Feferman, Israel Henrique Stokfisz; Fabrício, Lincoln Helder Zambaldi; Lorencini, Márcio
2013-01-01
BACKGROUND The sum of environmental and genetic factors affects the appearance and function of the skin as it ages. The identification of molecular changes that take place during skin aging provides biomarkers and possible targets for therapeutic intervention. Retinoic acid in different formulations has emerged as an alternative to prevent and repair age-related skin damage. OBJECTIVES To understand the effects of different retinoid formulations on the expression of genes associated with biological processes that undergo changes during skin aging. METHODS Ex-vivo skin samples were treated topically with different retinoid formulations. The modulation of biological processes associated with skin aging was measured by Reverse Transcription quantitative PCR (RT-qPCR). RESULTS A formulation containing microencapsulated retinol and a blend of active ingredients prepared as a triple nanoemulsion provided the best results for the modulation of biological, process-related genes that are usually affected during skin aging. CONCLUSION This association proved to be therapeutically more effective than tretinoin or microencapsulated retinol used singly. PMID:24474102
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Transmission electron microscopy in molecular structural biology: A historical survey.
Harris, J Robin
2015-09-01
In this personal, historic account of macromolecular transmission electron microscopy (TEM), published data from the 1940s through to recent times is surveyed, within the context of the remarkable progress that has been achieved during this time period. The evolution of present day molecular structural biology is described in relation to the associated biological disciplines. The contribution of numerous electron microscope pioneers to the development of the subject is discussed. The principal techniques for TEM specimen preparation, thin sectioning, metal shadowing, negative staining and plunge-freezing (vitrification) of thin aqueous samples are described, with a selection of published images to emphasise the virtues of each method. The development of digital image analysis and 3D reconstruction is described in detail as applied to electron crystallography and reconstructions from helical structures, 2D membrane crystals as well as single particle 3D reconstruction of icosahedral viruses and macromolecules. The on-going development of new software, algorithms and approaches is highlighted before specific examples of the historical progress of the structural biology of proteins and viruses are presented. Copyright © 2014 Elsevier Inc. All rights reserved.
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Integrated quantification and identification of aldehydes and ketones in biological samples.
Siegel, David; Meinema, Anne C; Permentier, Hjalmar; Hopfgartner, Gérard; Bischoff, Rainer
2014-05-20
The identification of unknown compounds remains to be a bottleneck of mass spectrometry (MS)-based metabolomics screening experiments. Here, we present a novel approach which facilitates the identification and quantification of analytes containing aldehyde and ketone groups in biological samples by adding chemical information to MS data. Our strategy is based on rapid autosampler-in-needle-derivatization with p-toluenesulfonylhydrazine (TSH). The resulting TSH-hydrazones are separated by ultrahigh-performance liquid chromatography (UHPLC) and detected by electrospray ionization-quadrupole-time-of-flight (ESI-QqTOF) mass spectrometry using a SWATH (Sequential Window Acquisition of all Theoretical Fragment-Ion Spectra) data-independent high-resolution mass spectrometry (HR-MS) approach. Derivatization makes small, poorly ionizable or retained analytes amenable to reversed phase chromatography and electrospray ionization in both polarities. Negatively charged TSH-hydrazone ions furthermore show a simple and predictable fragmentation pattern upon collision induced dissociation, which enables the chemo-selective screening for unknown aldehydes and ketones via a signature fragment ion (m/z 155.0172). By means of SWATH, targeted and nontargeted application scenarios of the suggested derivatization route are enabled in the frame of a single UHPLC-ESI-QqTOF-HR-MS workflow. The method's ability to simultaneously quantify and identify molecules containing aldehyde and ketone groups is demonstrated using 61 target analytes from various compound classes and a (13)C labeled yeast matrix. The identification of unknowns in biological samples is detailed using the example of indole-3-acetaldehyde.
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Kamali, Tschackad; Považay, Boris; Kumar, Sunil; Silberberg, Yaron; Hermann, Boris; Werkmeister, René; Drexler, Wolfgang; Unterhuber, Angelika
2014-10-01
We demonstrate a multimodal optical coherence tomography (OCT) and online Fourier transform coherent anti-Stokes Raman scattering (FTCARS) platform using a single sub-12 femtosecond (fs) Ti:sapphire laser enabling simultaneous extraction of structural and chemical ("morphomolecular") information of biological samples. Spectral domain OCT prescreens the specimen providing a fast ultrahigh (4×12 μm axial and transverse) resolution wide field morphologic overview. Additional complementary intrinsic molecular information is obtained by zooming into regions of interest for fast label-free chemical mapping with online FTCARS spectroscopy. Background-free CARS is based on a Michelson interferometer in combination with a highly linear piezo stage, which allows for quick point-to-point extraction of CARS spectra in the fingerprint region in less than 125 ms with a resolution better than 4 cm(-1) without the need for averaging. OCT morphology and CARS spectral maps indicating phosphate and carbonate bond vibrations from human bone samples are extracted to demonstrate the performance of this hybrid imaging platform.
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Solid phase sequencing of double-stranded nucleic acids
Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.
2002-01-01
This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.
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Boffetta, P; Islami, F
2013-04-01
The use of biological-based markers of exposure, intermediate effect, outcome, and susceptibility has become standard practice in cancer epidemiology, which has contributed to identification of several carcinogenic agents. Nevertheless, with the exception of biological agents, this contribution, in terms of providing sufficiently strong evidence as required by the International Agency for Research on Cancer (IARC) monographs, has been modest. We discuss the overall contribution of molecular epidemiology to identification of carcinogens, with focus on IARC monographs. For many carcinogens, valid biological markers of exposure and mechanisms of actions are not available. Molecular markers are usually assessed in single biological samples, which may not represent the actual exposure or biological events related to carcinogens. The contribution of molecular epidemiology to identification of carcinogens has mainly been limited to the carcinogens acting through a genotoxic mechanism, i.e. when carcinogens induce DNA damage. A number of factors, including certain hormones and overweight/obesity, may show carcinogenic effects through nongenotoxic pathways, for which mechanisms of carcinogenicity are not well identified and their biomarkers are sparse. Longitudinal assessment of biomarkers may provide more informative data in molecular epidemiology studies. For many carcinogens and mechanistic pathways, in particular nongenotoxic carcinogenicity, valid biological markers still need to be identified.
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Acoustical and optical radiation pressure and the development of single beam acoustical tweezers
NASA Astrophysics Data System (ADS)
Thomas, Jean-Louis; Marchiano, Régis; Baresch, Diego
2017-07-01
Studies on radiation pressure in acoustics and optics have enriched one another and have a long common history. Acoustic radiation pressure is used for metrology, levitation, particle trapping and actuation. However, the dexterity and selectivity of single-beam optical tweezers are still to be matched with acoustical devices. Optical tweezers can trap, move and position micron size particles, biological samples or even atoms with subnanometer accuracy in three dimensions. One limitation of optical tweezers is the weak force that can be applied without thermal damage due to optical absorption. Acoustical tweezers overcome this limitation since the radiation pressure scales as the field intensity divided by the speed of propagation of the wave. However, the feasibility of single beam acoustical tweezers was demonstrated only recently. In this paper, we propose a historical review of the strong similarities but also the specificities of acoustical and optical radiation pressures, from the expression of the force to the development of single-beam acoustical tweezers.
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NASA Astrophysics Data System (ADS)
Trusiak, Maciej; Micó, Vicente; Patorski, Krzysztof; García-Monreal, Javier; Sluzewski, Lukasz; Ferreira, Carlos
2016-08-01
In this contribution we propose two Hilbert-Huang Transform based algorithms for fast and accurate single-shot and two-shot quantitative phase imaging applicable in both on-axis and off-axis configurations. In the first scheme a single fringe pattern containing information about biological phase-sample under study is adaptively pre-filtered using empirical mode decomposition based approach. Further it is phase demodulated by the Hilbert Spiral Transform aided by the Principal Component Analysis for the local fringe orientation estimation. Orientation calculation enables closed fringes efficient analysis and can be avoided using arbitrary phase-shifted two-shot Gram-Schmidt Orthonormalization scheme aided by Hilbert-Huang Transform pre-filtering. This two-shot approach is a trade-off between single-frame and temporal phase shifting demodulation. Robustness of the proposed techniques is corroborated using experimental digital holographic microscopy studies of polystyrene micro-beads and red blood cells. Both algorithms compare favorably with the temporal phase shifting scheme which is used as a reference method.
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Single-cell genomic profiling of acute myeloid leukemia for clinical use: A pilot study
Yan, Benedict; Hu, Yongli; Ban, Kenneth H.K.; Tiang, Zenia; Ng, Christopher; Lee, Joanne; Tan, Wilson; Chiu, Lily; Tan, Tin Wee; Seah, Elaine; Ng, Chin Hin; Chng, Wee-Joo; Foo, Roger
2017-01-01
Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable. PMID:28454300
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Sensing of Streptococcus mutans by microscopic imaging ellipsometry
NASA Astrophysics Data System (ADS)
Khaleel, Mai Ibrahim; Chen, Yu-Da; Chien, Ching-Hang; Chang, Yia-Chung
2017-05-01
Microscopic imaging ellipsometry is an optical technique that uses an objective and sensing procedure to measure the ellipsometric parameters Ψ and Δ in the form of microscopic maps. This technique is well known for being noninvasive and label-free. Therefore, it can be used to detect and characterize biological species without any impact. Microscopic imaging ellipsometry was used to measure the optical response of dried Streptococcus mutans cells on a glass substrate. The ellipsometric Ψ and Δ maps were obtained with the Optrel Multiskop system for specular reflection in the visible range (λ=450 to 750 nm). The Ψ and Δ images at 500, 600, and 700 nm were analyzed using three different theoretical models with single-bounce, two-bounce, and multibounce light paths to obtain the optical constants and height distribution. The obtained images of the optical constants show different aspects when comparing the single-bounce analysis with the two-bounce or multibounce analysis in detecting S. mutans samples. Furthermore, the height distributions estimated by two-bounce and multibounce analyses of S. mutans samples were in agreement with the thickness values measured by AFM, which implies that the two-bounce and multibounce analyses can provide information complementary to that obtained by a single-bounce light path.
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On-chip Magnetic Separation and Cell Encapsulation in Droplets†
Chen, Aaron; Byvank, Tom; Chang, Woo-Jin; Bharde, Atul; Vieira, Greg; Miller, Brandon; Chalmers, Jeffrey J.; Bashir, Rashid; Sooryakumar, Ratnasingham
2014-01-01
The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for myriad of cell analyses over conventional methods, which require bulk samples providing only averaged information on cell metabolism. We report on a device that integrates mobile magnetic trap array with microfluidic technology to provide, combined functionality of separation of immunomagnetically labeled cells or magnetic beads and their encapsulation with reagents into pico-liter droplets. This scheme of simultaneous reagent delivery and compartmentalization of the cells immediately after sorting, all performed seamlessly within the same chip, offers unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use and freshness of the reagent solution that reacts only with separated objects, and tunable encapsulation characteristics independent of the input flow. In addition to the demonstrated preliminary cell viability assay, the device can potentially be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool. PMID:23370785
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Liu, Xiao-Lin; Liu, Wen-Jun
2007-04-01
Analyses of microbial community structure in bio-ceramics (BC) and biological activated carbon (BAC), which widely used in drinking water treatment were performed by polymerase-chain-reaction-single-strand-conformation-polymorphism (PCR-SSCP) targeted eubacterial 16S ribosomal RNA gene. Microorganisms on bio-ceramics and biological activated carbon were detached by ultrasonic, culturing on R2A and LB agar, respectively, followed by genome DNA extracting. Results show that larger than 10 kb genome DNA could be extracted from all the samples except the BAC samples processed by ultrasonic. However, quantities of the extracted DNA were different. 408 bp gene fragments were observed after PCR using the extracted genome DNA as templates. These gene fragments were digested with lambda exonuclease followed by SSCP electrophoresis. Same SSCP profiles were observed between ultrasonic eluting, R2A and LB agar culturing. The identity of the segment from bio-ceramics with uncultured Pseudomonas sp. Clone FTL201 16S rDNA (GenBank, AF509293.1) fragment was 92%, and identities of the two segments from BAC with Bacillus sp. JH19 16S rDNA (GenBank , DQ232748.1) fragment and Bacterium VA-S-11 16S rDNA (GenBank, AY395279.1) fragment were 100% and 99%, respectively.
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Mungwira, Randy G; Nyangulu, Wongani; Misiri, James; Iphani, Steven; Ng'ong'ola, Ruby; Chirambo, Chawanangwa M; Masiye, Francis; Mfutso-Bengo, Joseph
2015-12-02
This paper discusses the contentious issue of reuse of stored biological samples and data obtained from research participants in past clinical research to answer future ethical and scientifically valid research questions. Many countries have regulations and guidelines that guide the use and exportation of stored biological samples and data. However, there are variations in regulations and guidelines governing the reuse of stored biological samples and data in Sub-Saharan Africa including Malawi. The current research ethics regulations and guidelines in Malawi do not allow indefinite storage and reuse of biological samples and data for future unspecified research. This comes even though the country has managed to answer pertinent research questions using stored biological samples and data. We acknowledge the limited technical expertise and equipment unavailable in Malawi that necessitates exportation of biological samples and data and the genuine concern raised by the regulatory authorities about the possible exploitation of biological samples and data by researchers. We also acknowledge that Malawi does not have bio-banks for storing biological samples and data for future research purposes. This creates room for possible exploitation of biological samples and data collected from research participants in primary research projects in Malawi. However, research ethics committees require completion and approval of material transfer agreements and data transfer agreements for biological samples and data collected for research purposes respectively and this requirement may partly address the concern raised by the regulatory authorities. Our concern though is that there is no such requirement for biological samples and data collected from patients for clinical or diagnostic purposes. In conclusion, we propose developing a medical data and material transfer agreement for biological samples and data collected from patients for clinical or diagnostic purposes in both public and private health facilities that may end up in research centers outside Malawi. We also propose revision of the current research ethics regulations and guidelines in Malawi in order to allow secondary use of biological samples and data collected from primary research projects as a way of maximizing the use of collected samples and data. Finally, we call for consultation of all stakeholders within the Malawi research community when regulatory authorities are developing policies that govern research in Malawi.
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Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader.
Schaefer, Jorrit; Jovanovic, Goran; Kotta-Loizou, Ioly; Buck, Martin
2016-06-15
Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene). Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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Imaging proteins at the single-molecule level.
Longchamp, Jean-Nicolas; Rauschenbach, Stephan; Abb, Sabine; Escher, Conrad; Latychevskaia, Tatiana; Kern, Klaus; Fink, Hans-Werner
2017-02-14
Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.
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Highly multiplexed subcellular RNA sequencing in situ
Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Yang, Joyce L.; Terry, Richard; Jeanty, Sauveur S. F.; Li, Chao; Amamoto, Ryoji; Peters, Derek T.; Turczyk, Brian M.; Marblestone, Adam H.; Inverso, Samuel A.; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.
2014-01-01
Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. PMID:24578530
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Imaging proteins at the single-molecule level
Longchamp, Jean-Nicolas; Rauschenbach, Stephan; Abb, Sabine; Escher, Conrad; Latychevskaia, Tatiana; Kern, Klaus; Fink, Hans-Werner
2017-01-01
Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process. PMID:28087691
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Rotational manipulation of single cells and organisms using acoustic waves
Ahmed, Daniel; Ozcelik, Adem; Bojanala, Nagagireesh; Nama, Nitesh; Upadhyay, Awani; Chen, Yuchao; Hanna-Rose, Wendy; Huang, Tony Jun
2016-01-01
The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation. PMID:27004764
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Single-protein detection in crowded molecular environments in cryo-EM images
Rickgauer, J Peter; Grigorieff, Nikolaus; Denk, Winfried
2017-01-01
We present an approach to study macromolecular assemblies by detecting component proteins’ characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and—in the presence of protein background—a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material. DOI: http://dx.doi.org/10.7554/eLife.25648.001 PMID:28467302
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Single shot near edge x-ray absorption fine structure spectroscopy in the laboratory
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mantouvalou, I., E-mail: ioanna.mantouvalou@tu-berlin.de; Witte, K.; Martyanov, W.
With the help of adapted off-axis reflection zone plates, near edge X-ray absorption fine structure spectra at the C and N K-absorption edge have been recorded using a single 1.2 ns long soft X-ray pulse. The transmission experiments were performed with a laser-produced plasma source in the laboratory rendering time resolved measurements feasible independent on large scale facilities. A resolving power of E/ΔE ∼ 950 at the respective edges could be demonstrated. A comparison of single shot spectra with those collected with longer measuring time proves that all features of the used reference samples (silicon nitrate and polyimide) can be resolved in 1.2 ns.more » Hence, investigations of radiation sensitive biological specimen become possible due to the high efficiency of the optical elements enabling low dose experiments.« less
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Single-sided magnetic resonance profiling in biological and materials science.
Danieli, Ernesto; Blümich, Bernhard
2013-04-01
Single-sided NMR was inspired by the oil industry that strived to improve the performance of well-logging tools to measure the properties of fluids confined downhole. This unconventional way of implementing NMR, in which stray magnetic and radio frequency fields are used to recover information of arbitrarily large objects placed outside the magnet, motivated the development of handheld NMR sensors. These devices have moved the technique to different scientific disciplines. The current work gives a review of the most relevant magnets and methodologies developed to generate NMR information from spatially localized regions of samples placed in close proximity to the sensors. When carried out systematically, such measurements lead to 'single-sided depth profiles' or one-dimensional images. This paper presents recent and most relevant applications as well as future perspectives of this growing branch of MRI. Copyright © 2012 Elsevier Inc. All rights reserved.
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Rotational manipulation of single cells and organisms using acoustic waves.
Ahmed, Daniel; Ozcelik, Adem; Bojanala, Nagagireesh; Nama, Nitesh; Upadhyay, Awani; Chen, Yuchao; Hanna-Rose, Wendy; Huang, Tony Jun
2016-03-23
The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation.
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Automated DBS microsampling, microscale automation and microflow LC-MS for therapeutic protein PK.
Zhang, Qian; Tomazela, Daniela; Vasicek, Lisa A; Spellman, Daniel S; Beaumont, Maribel; Shyong, BaoJen; Kenny, Jacqueline; Fauty, Scott; Fillgrove, Kerry; Harrelson, Jane; Bateman, Kevin P
2016-04-01
Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS. We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold. Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.
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Chan, Yvonne L; Schanzenbach, David; Hickerson, Michael J
2014-09-01
Methods that integrate population-level sampling from multiple taxa into a single community-level analysis are an essential addition to the comparative phylogeographic toolkit. Detecting how species within communities have demographically tracked each other in space and time is important for understanding the effects of future climate and landscape changes and the resulting acceleration of extinctions, biological invasions, and potential surges in adaptive evolution. Here, we present a statistical framework for such an analysis based on hierarchical approximate Bayesian computation (hABC) with the goal of detecting concerted demographic histories across an ecological assemblage. Our method combines population genetic data sets from multiple taxa into a single analysis to estimate: 1) the proportion of a community sample that demographically expanded in a temporally clustered pulse and 2) when the pulse occurred. To validate the accuracy and utility of this new approach, we use simulation cross-validation experiments and subsequently analyze an empirical data set of 32 avian populations from Australia that are hypothesized to have expanded from smaller refugia populations in the late Pleistocene. The method can accommodate data set heterogeneity such as variability in effective population size, mutation rates, and sample sizes across species and exploits the statistical strength from the simultaneous analysis of multiple species. This hABC framework used in a multitaxa demographic context can increase our understanding of the impact of historical climate change by determining what proportion of the community responded in concert or independently and can be used with a wide variety of comparative phylogeographic data sets as biota-wide DNA barcoding data sets accumulate. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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A comparison of machine learning and Bayesian modelling for molecular serotyping.
Newton, Richard; Wernisch, Lorenz
2017-08-11
Streptococcus pneumoniae is a human pathogen that is a major cause of infant mortality. Identifying the pneumococcal serotype is an important step in monitoring the impact of vaccines used to protect against disease. Genomic microarrays provide an effective method for molecular serotyping. Previously we developed an empirical Bayesian model for the classification of serotypes from a molecular serotyping array. With only few samples available, a model driven approach was the only option. In the meanwhile, several thousand samples have been made available to us, providing an opportunity to investigate serotype classification by machine learning methods, which could complement the Bayesian model. We compare the performance of the original Bayesian model with two machine learning algorithms: Gradient Boosting Machines and Random Forests. We present our results as an example of a generic strategy whereby a preliminary probabilistic model is complemented or replaced by a machine learning classifier once enough data are available. Despite the availability of thousands of serotyping arrays, a problem encountered when applying machine learning methods is the lack of training data containing mixtures of serotypes; due to the large number of possible combinations. Most of the available training data comprises samples with only a single serotype. To overcome the lack of training data we implemented an iterative analysis, creating artificial training data of serotype mixtures by combining raw data from single serotype arrays. With the enhanced training set the machine learning algorithms out perform the original Bayesian model. However, for serotypes currently lacking sufficient training data the best performing implementation was a combination of the results of the Bayesian Model and the Gradient Boosting Machine. As well as being an effective method for classifying biological data, machine learning can also be used as an efficient method for revealing subtle biological insights, which we illustrate with an example.
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Unravelling biology and shifting paradigms in cancer with single-cell sequencing.
Baslan, Timour; Hicks, James
2017-08-24
The fundamental operative unit of a cancer is the genetically and epigenetically innovative single cell. Whether proliferating or quiescent, in the primary tumour mass or disseminated elsewhere, single cells govern the parameters that dictate all facets of the biology of cancer. Thus, single-cell analyses provide the ultimate level of resolution in our quest for a fundamental understanding of this disease. Historically, this quest has been hampered by technological shortcomings. In this Opinion article, we argue that the rapidly evolving field of single-cell sequencing has unshackled the cancer research community of these shortcomings. From furthering an elemental understanding of intra-tumoural genetic heterogeneity and cancer genome evolution to illuminating the governing principles of disease relapse and metastasis, we posit that single-cell sequencing promises to unravel the biology of all facets of this disease.
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Extracting physics of life at the molecular level: A review of single-molecule data analyses.
Colomb, Warren; Sarkar, Susanta K
2015-06-01
Studying individual biomolecules at the single-molecule level has proved very insightful recently. Single-molecule experiments allow us to probe both the equilibrium and nonequilibrium properties as well as make quantitative connections with ensemble experiments and equilibrium thermodynamics. However, it is important to be careful about the analysis of single-molecule data because of the noise present and the lack of theoretical framework for processes far away from equilibrium. Biomolecular motion, whether it is free in solution, on a substrate, or under force, involves thermal fluctuations in varying degrees, which makes the motion noisy. In addition, the noise from the experimental setup makes it even more complex. The details of biologically relevant interactions, conformational dynamics, and activities are hidden in the noisy single-molecule data. As such, extracting biological insights from noisy data is still an active area of research. In this review, we will focus on analyzing both fluorescence-based and force-based single-molecule experiments and gaining biological insights at the single-molecule level. Inherently nonequilibrium nature of biological processes will be highlighted. Simulated trajectories of biomolecular diffusion will be used to compare and validate various analysis techniques. Copyright © 2015 Elsevier B.V. All rights reserved.
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Hanson, E; Ingold, S; Haas, C; Ballantyne, J
2018-05-01
The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and inter-sample differential gene expression revealed by agglomerative hierarchical clustering. Copyright © 2018 Elsevier B.V. All rights reserved.
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Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.
2015-01-01
Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316
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Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G
2015-05-01
Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.
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A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection
Ng, Siemon H. S.; Vandeputte, Olivier; Aljanahi, Aisha; Deyati, Avisek; Cassart, Jean-Pol; Charlebois, Robert L.; Taliaferro, Lanyn P.
2017-01-01
ABSTRACT The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. IMPORTANCE Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms. PMID:28932815
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Zarzycki, Paweł K; Slączka, Magdalena M; Zarzycka, Magdalena B; Bartoszuk, Małgorzata A; Włodarczyk, Elżbieta; Baran, Michał J
2011-11-01
This paper is a continuation of our previous research focusing on development of micro-TLC methodology under temperature-controlled conditions. The main goal of present paper is to demonstrate separation and detection capability of micro-TLC technique involving simple analytical protocols without multi-steps sample pre-purification. One of the advantages of planar chromatography over its column counterpart is that each TLC run can be performed using non-previously used stationary phase. Therefore, it is possible to fractionate or separate complex samples characterized by heavy biological matrix loading. In present studies components of interest, mainly steroids, were isolated from biological samples like fish bile using single pre-treatment steps involving direct organic liquid extraction and/or deproteinization by freeze-drying method. Low-molecular mass compounds with polarity ranging from estetrol to progesterone derived from the environmental samples (lake water, untreated and treated sewage waters) were concentrated using optimized solid-phase extraction (SPE). Specific bands patterns for samples derived from surface water of the Middle Pomerania in northern part of Poland can be easily observed on obtained micro-TLC chromatograms. This approach can be useful as simple and non-expensive complementary method for fast control and screening of treated sewage water discharged by the municipal wastewater treatment plants. Moreover, our experimental results show the potential of micro-TLC as an efficient tool for retention measurements of a wide range of steroids under reversed-phase (RP) chromatographic conditions. These data can be used for further optimalization of SPE or HPLC systems working under RP conditions. Furthermore, we also demonstrated that micro-TLC based analytical approach can be applied as an effective method for the internal standard (IS) substance search. Generally, described methodology can be applied for fast fractionation or screening of the whole range of target substances as well as chemo-taxonomic studies and fingerprinting of complex mixtures, which are present in biological or environmental samples. Due to low consumption of eluent (usually 0.3-1mL/run) mainly composed of water-alcohol binary mixtures, this method can be considered as environmentally friendly and green chemistry focused analytical tool, supplementary to analytical protocols involving column chromatography or planar micro-fluidic devices. Copyright © 2011 Elsevier Ltd. All rights reserved.
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The technology and biology of single-cell RNA sequencing.
Kolodziejczyk, Aleksandra A; Kim, Jong Kyoung; Svensson, Valentine; Marioni, John C; Teichmann, Sarah A
2015-05-21
The differences between individual cells can have profound functional consequences, in both unicellular and multicellular organisms. Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells. Already, single-cell RNA-sequencing methods have revealed new biology in terms of the composition of tissues, the dynamics of transcription, and the regulatory relationships between genes. Rapid technological developments at the level of cell capture, phenotyping, molecular biology, and bioinformatics promise an exciting future with numerous biological and medical applications. Copyright © 2015 Elsevier Inc. All rights reserved.
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A Novel Technique for Sterilization Using a Power Self-Regulated Single-Mode Microwave Cavity.
Reverte-Ors, Juan D; Pedreño-Molina, Juan L; Fernández, Pablo S; Lozano-Guerrero, Antonio J; Periago, Paula M; Díaz-Morcillo, Alejandro
2017-06-07
In this paper, a novel technique to achieve precise temperatures in food sterilization has been proposed. An accurate temperature profile is needed in order to reach a commitment between the total removal of pathogens inside the product and the preservation of nutritional and organoleptic characteristics. The minimal variation of the target temperature in the sample by means of a monitoring and control software platform, allowing temperature stabilization over 100 °C, is the main goal of this work. A cylindrical microwave oven, under pressure conditions and continuous control of the microwave supply power as function of the final temperature inside the sample, has been designed and developed with conditions of single-mode resonance. The uniform heating in the product is achieved by means of sample movement and the self-regulated power control using the measured temperature. Finally, for testing the sterilization of food with this technology, specific biological validation based on Bacillus cereus as a biosensor of heat inactivation has been incorporated as a distribution along the sample in the experimental process to measure the colony-forming units (CFUs) for different food samples (laboratory medium, soup, or fish-based animal by-products). The obtained results allow the validation of this new technology for food sterilization with precise control of the microwave system to ensure the uniform elimination of pathogens using high temperatures.
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A Novel Technique for Sterilization Using a Power Self-Regulated Single-Mode Microwave Cavity
Reverte-Ors, Juan D.; Pedreño-Molina, Juan L.; Fernández, Pablo S.; Lozano-Guerrero, Antonio J.; Periago, Paula M.; Díaz-Morcillo, Alejandro
2017-01-01
In this paper, a novel technique to achieve precise temperatures in food sterilization has been proposed. An accurate temperature profile is needed in order to reach a commitment between the total removal of pathogens inside the product and the preservation of nutritional and organoleptic characteristics. The minimal variation of the target temperature in the sample by means of a monitoring and control software platform, allowing temperature stabilization over 100 °C, is the main goal of this work. A cylindrical microwave oven, under pressure conditions and continuous control of the microwave supply power as function of the final temperature inside the sample, has been designed and developed with conditions of single-mode resonance. The uniform heating in the product is achieved by means of sample movement and the self-regulated power control using the measured temperature. Finally, for testing the sterilization of food with this technology, specific biological validation based on Bacillus cereus as a biosensor of heat inactivation has been incorporated as a distribution along the sample in the experimental process to measure the colony-forming units (CFUs) for different food samples (laboratory medium, soup, or fish-based animal by-products). The obtained results allow the validation of this new technology for food sterilization with precise control of the microwave system to ensure the uniform elimination of pathogens using high temperatures. PMID:28590423
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Cell fixation and preservation for droplet-based single-cell transcriptomics.
Alles, Jonathan; Karaiskos, Nikos; Praktiknjo, Samantha D; Grosswendt, Stefanie; Wahle, Philipp; Ruffault, Pierre-Louis; Ayoub, Salah; Schreyer, Luisa; Boltengagen, Anastasiya; Birchmeier, Carmen; Zinzen, Robert; Kocks, Christine; Rajewsky, Nikolaus
2017-05-19
Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. By using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.
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NASA Astrophysics Data System (ADS)
McMillan, N. J.; Chavez, A.; Chanover, N.; Voelz, D.; Uckert, K.; Tawalbeh, R.; Gariano, J.; Dragulin, I.; Xiao, X.; Hull, R.
2014-12-01
Rapid, in-situ methods for identification of biologic and non-biologic mineral precipitation sites permit mapping of biological hot spots. Two portable spectrometers, Laser-Induced Breakdown Spectroscopy (LIBS) and Acoustic-Optic Tunable Filter Reflectance Spectroscopy (AOTFRS) were used to differentiate between bacterially influenced and inorganically precipitated calcite specimens from Fort Stanton Cave, NM, USA. LIBS collects light emitted from the decay of excited electrons in a laser ablation plasma; the spectrum is a chemical fingerprint of the analyte. AOTFRS collects light reflected from the surface of a specimen and provides structural information about the material (i.e., the presence of O-H bonds). These orthogonal data sets provide a rigorous method to determine the origin of calcite in cave deposits. This study used a set of 48 calcite samples collected from Fort Stanton cave. Samples were examined in SEM for the presence of biologic markers; these data were used to separate the samples into biologic and non-biologic groups. Spectra were modeled using the multivariate technique Partial Least Squares Regression (PLSR). Half of the spectra were used to train a PLSR model, in which biologic samples were assigned to the independent variable "0" and non-biologic samples were assigned the variable "1". Values of the independent variable were calculated for each of the training samples, which were close to 0 for the biologic samples (-0.09 - 0.23) and close to 1 for the non-biologic samples (0.57 - 1.14). A Value of Apparent Distinction (VAD) of 0.55 was used to numerically distinguish between the two groups; any sample with an independent variable value < 0.55 was classified as having a biologic origin; a sample with a value > 0.55 was determined to be non-biologic in origin. After the model was trained, independent variable values for the remaining half of the samples were calculated. Biologic or non-biologic origin was assigned by comparison to the VAD. Using LIBS data alone, the model has a 92% success rate, correctly identifying 23 of 25 samples. Modeling of AOTFRS spectra and the combined LIBS-AOTFRS data set have similar success rates. This study demonstrates that rapid, portable LIBS and AOTFRS instruments can be used to map the spatial distribution of biologic precipitation in caves.
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Sengupta, Debanti; Kim, Tae Jin; Almasi, Sepideh; Miller, Stuart; Marton, Zsolt; Nagarkar, Vivek; Pratx, Guillem
2018-04-16
Radioluminescence microscopy is an emerging modality that can be used to image radionuclide probes with micron-scale resolution. This technique is particularly useful as a way to probe the metabolic behavior of single cells and to screen and characterize radiopharmaceuticals, but the quality of the images is critically dependent on the scintillator material used to image the cells. In this paper, we detail the development of a microscopy dish made of a thin-film scintillating material, Lu2O3:Eu, that could be used as the blueprint for a future consumable product. After developing a simple quality control method based on long-lived alpha and beta sources, we characterize the radioluminescence properties of various thin-film scintillator samples. We find consistent performance for most samples, but also identify a few samples that do not meet the specifications, thus stressing the need for routine quality control prior to biological experiments. In addition, we test and quantify the transparency of the material, and demonstrate that transparency correlates with thickness. Finally, we evaluate the biocompatibility of the material and show that the microscopy dish can produce radioluminescent images of live single cells.
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Surface plasmon resonance biosensors for highly sensitive detection in real samples
NASA Astrophysics Data System (ADS)
Sepúlveda, B.; Carrascosa, L. G.; Regatos, D.; Otte, M. A.; Fariña, D.; Lechuga, L. M.
2009-08-01
In this work we summarize the main results obtained with the portable surface plasmon resonance (SPR) device developed in our group (commercialised by SENSIA, SL, Spain), highlighting its applicability for the real-time detection of extremely low concentrations of toxic pesticides in environmental water samples. In addition, we show applications in clinical diagnosis as, on the one hand, the real-time and label-free detection of DNA hybridization and single point mutations at the gene BRCA-1, related to the predisposition in women to develop an inherited breast cancer and, on the other hand, the analysis of protein biomarkers in biological samples (urine, serum) for early detection of diseases. Despite the large number of applications already proven, the SPR technology has two main drawbacks: (i) not enough sensitivity for some specific applications (where pM-fM or single-molecule detection are needed) (ii) low multiplexing capabilities. In order solve such drawbacks, we work in several alternative configurations as the Magneto-optical Surface Plasmon Resonance sensor (MOSPR) based on a combination of magnetooptical and ferromagnetic materials, to improve the SPR sensitivity, or the Localized Surface Plasmon Resonance (LSPR) based on nanostructures (nanoparticles, nanoholes,...), for higher multiplexing capabilities.
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Handschuh, S; Beisser, C J; Ruthensteiner, B; Metscher, B D
2017-07-01
Dual-energy computed tomography (DECT) uses two different x-ray energy spectra in order to differentiate between tissues, materials or elements in a single sample or patient. DECT is becoming increasingly popular in clinical imaging and preclinical in vivo imaging of small animal models, but there have been only very few reports on ex vivo DECT of biological samples at microscopic resolutions. The present study has three main aims. First, we explore the potential of microscopic DECT (microDECT) for delivering isotropic multichannel 3D images of fixed biological samples with standard commercial laboratory-based microCT setups at spatial resolutions reaching below 10 μm. Second, we aim for retaining the maximum image resolution and quality during the material decomposition. Third, we want to test the suitability for microDECT imaging of different contrast agents currently used for ex vivo staining of biological samples. To address these aims, we used microCT scans of four different samples stained with x-ray dense contrast agents. MicroDECT scans were acquired with five different commercial microCT scanners from four companies. We present a detailed description of the microDECT workflow, including sample preparation, image acquisition, image processing and postreconstruction material decomposition, which may serve as practical guide for applying microDECT. The MATLAB script (The Mathworks Inc., Natick, MA, USA) used for material decomposition (including a graphical user interface) is provided as a supplement to this paper (https://github.com/microDECT/DECTDec). In general, the presented microDECT workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in the separate material fractions after basis material decomposition. In addition to decomposition of mineralized tissues (inherent sample contrast) and stained soft tissues, we present a case of double labelling of different soft tissues with subsequent material decomposition. We conclude that, in contrast to in vivo DECT examinations, small ex vivo specimens offer some clear advantages regarding technical parameters of the microCT setup and the use of contrast agents. These include a higher flexibility in source peak voltages and x-ray filters, a lower degree of beam hardening due to small sample size, the lack of restriction to nontoxic contrast agents and the lack of a limit in exposure time and radiation dose. We argue that microDECT, because of its flexibility combined with already established contrast agents and the vast number of currently unexploited stains, will in future represent an important technique for various applications in biological research. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.
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Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.
Geng, Tao; Mathies, Richard A
2015-01-01
The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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The low single nucleotide polymorphism heritability of plasma and saliva cortisol levels.
Neumann, Alexander; Direk, Nese; Crawford, Andrew A; Mirza, Saira; Adams, Hieab; Bolton, Jennifer; Hayward, Caroline; Strachan, David P; Payne, Erin K; Smith, Jennifer A; Milaneschi, Yuri; Penninx, Brenda; Hottenga, Jouke J; de Geus, Eco; Oldehinkel, Albertine J; van der Most, Peter J; de Rijke, Yolanda; Walker, Brian R; Tiemeier, Henning
2017-11-01
Cortisol is an important stress hormone affected by a variety of biological and environmental factors, such as the circadian rhythm, exercise and psychological stress. Cortisol is mostly measured using blood or saliva samples. A number of genetic variants have been found to contribute to cortisol levels with these methods. While the effects of several specific single genetic variants is known, the joint genome-wide contribution to cortisol levels is unclear. Our aim was to estimate the amount of cortisol variance explained by common single nucleotide polymorphisms, i.e. the SNP heritability, using a variety of cortisol measures, cohorts and analysis approaches. We analyzed morning plasma (n=5705) and saliva levels (n=1717), as well as diurnal saliva levels (n=1541), in the Rotterdam Study using genomic restricted maximum likelihood estimation. Additionally, linkage disequilibrium score regression was fitted on the results of genome-wide association studies (GWAS) performed by the CORNET consortium on morning plasma cortisol (n=12,597) and saliva cortisol (n=7703). No significant SNP heritability was detected for any cortisol measure, sample or analysis approach. Point estimates ranged from 0% to 9%. Morning plasma cortisol in the CORNET cohorts, the sample with the most power, had a 6% [95%CI: 0-13%] SNP heritability. The results consistently suggest a low SNP heritability of these acute and short-term measures of cortisol. The low SNP heritability may reflect the substantial environmental and, in particular, situational component of these cortisol measures. Future GWAS will require very large sample sizes. Alternatively, more long-term cortisol measures such as hair cortisol samples are needed to discover further genetic pathways regulating cortisol concentrations. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Strong-Gunderson, Janet M.; Palumbo, Anthony V.
1998-01-01
The present invention is a biological tracer method for characterizing the movement of a material through a medium, comprising the steps of: introducing a biological tracer comprising a microorganism having ice nucleating activity into a medium; collecting at least one sample of the medium from a point removed from the introduction point; and analyzing the sample for the presence of the biological tracer. The present invention is also a method for using a biological tracer as a label for material identification by introducing a biological tracer having ice nucleating activity into a material, collecting a sample of a portion of the labelled material and analyzing the sample for the presence of the biological tracer.
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Strong-Gunderson, J.M.; Palumbo, A.V.
1998-09-15
The present invention is a biological tracer method for characterizing the movement of a material through a medium, comprising the steps of: introducing a biological tracer comprising a microorganism having ice nucleating activity into a medium; collecting at least one sample of the medium from a point removed from the introduction point; and analyzing the sample for the presence of the biological tracer. The present invention is also a method for using a biological tracer as a label for material identification by introducing a biological tracer having ice nucleating activity into a material, collecting a sample of a portion of the labelled material and analyzing the sample for the presence of the biological tracer. 2 figs.
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Seok, Junhee; Davis, Ronald W; Xiao, Wenzhong
2015-01-01
Accumulated biological knowledge is often encoded as gene sets, collections of genes associated with similar biological functions or pathways. The use of gene sets in the analyses of high-throughput gene expression data has been intensively studied and applied in clinical research. However, the main interest remains in finding modules of biological knowledge, or corresponding gene sets, significantly associated with disease conditions. Risk prediction from censored survival times using gene sets hasn't been well studied. In this work, we propose a hybrid method that uses both single gene and gene set information together to predict patient survival risks from gene expression profiles. In the proposed method, gene sets provide context-level information that is poorly reflected by single genes. Complementarily, single genes help to supplement incomplete information of gene sets due to our imperfect biomedical knowledge. Through the tests over multiple data sets of cancer and trauma injury, the proposed method showed robust and improved performance compared with the conventional approaches with only single genes or gene sets solely. Additionally, we examined the prediction result in the trauma injury data, and showed that the modules of biological knowledge used in the prediction by the proposed method were highly interpretable in biology. A wide range of survival prediction problems in clinical genomics is expected to benefit from the use of biological knowledge.
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Seok, Junhee; Davis, Ronald W.; Xiao, Wenzhong
2015-01-01
Accumulated biological knowledge is often encoded as gene sets, collections of genes associated with similar biological functions or pathways. The use of gene sets in the analyses of high-throughput gene expression data has been intensively studied and applied in clinical research. However, the main interest remains in finding modules of biological knowledge, or corresponding gene sets, significantly associated with disease conditions. Risk prediction from censored survival times using gene sets hasn’t been well studied. In this work, we propose a hybrid method that uses both single gene and gene set information together to predict patient survival risks from gene expression profiles. In the proposed method, gene sets provide context-level information that is poorly reflected by single genes. Complementarily, single genes help to supplement incomplete information of gene sets due to our imperfect biomedical knowledge. Through the tests over multiple data sets of cancer and trauma injury, the proposed method showed robust and improved performance compared with the conventional approaches with only single genes or gene sets solely. Additionally, we examined the prediction result in the trauma injury data, and showed that the modules of biological knowledge used in the prediction by the proposed method were highly interpretable in biology. A wide range of survival prediction problems in clinical genomics is expected to benefit from the use of biological knowledge. PMID:25933378
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Opit, George P; Perret, Jamis; Holt, Kiffnie; Nechols, James R; Margolies, David C; Williams, Kimberly A
2009-02-01
Efficacy, costs, and impact on crop salability of various biological and chemical control strategies for Tetranychus urticae Koch (Acari: Tetranychidae) were evaluated on mixed plantings of impatiens, Impatiens wallerana Hook.f (Ericales: Balsaminaceae), and ivy geranium, Pelargonium peltatum (1.) L'Hér. Ex Aiton (Geraniales: Geraniaceae), cultivars in commercial greenhouses. Chemical control consisting of the miticide bifenazate (Floramite) was compared with two biological control strategies using the predatory mite Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae). Treatments were 1) a single, early application of bifenazate; 2) a single, early release of predatory mites at a 1:4 predator:pest ratio based on leaf samples to estimate pest density; 3) a weekly release of predatory mites at numbers based on the area covered by the crop; and 4) an untreated control. T. urticae populations were monitored for 3 wk after the earliest treatment. When plants were ready for market, their salability was estimated. Bifenazate and density-based P. persimilis treatments effectively reduced T. urticae numbers starting 1 wk after plants had been treated, whereas the scheduled, area-based P. persimilis treatment had little or no effect. The percentage of flats that could be sold at the highest market wholesale price ranged from 15 to 33%, 44 to 86%, 84 to 95%, and 92 to 100%, in the control, weekly area-based P. persimilis, bifenazate, and single density-based P. persimilis treatments, respectively. We have shown that in commercial greenhouse production of herbaceous ornamental bedding plants, estimating pest density to determine the appropriate number of predators to release is as effective and offers nearly the same economic benefit as prophylactic use of pesticides.
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Single-Molecule Counting of Point Mutations by Transient DNA Binding
NASA Astrophysics Data System (ADS)
Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan
2017-03-01
High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.
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Family structure, victimization, and child mental health in a nationally representative sample.
Turner, Heather A; Finkelhor, David; Hamby, Sherry L; Shattuck, Anne
2013-06-01
Utilizing the 2008 National Survey of Children's Exposure to Violence (NatSCEV), the current study compares past year rates of 7 forms of child victimization (maltreatment, assault, peer victimization, property crime, witnessing family violence and exposure to community violence) across 3 different family structure types (two biological/adoptive parents, single parent, step/cohabiting family) among a representative sample of 4046 U.S. children ages 2-17. The study also considers whether certain social-contextual risk factors help to explain family structure variations in victimization, and the extent to which victimization exposure accounts for family structure differences in distress symptom levels. Findings showed significantly elevated rates of almost all types of victimization among children in both nontraditional family types, relative to those living with two biological/adoptive parents. Factors associated with increased victimization risk in these families include high parental conflict, drug or alcohol problems, family adversity, and community disorder. A summary measure of children's exposure to multiple forms of victimization was the strongest predictor of distress symptoms. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Mass Spectrometric Imaging Using Laser Ablation and Solvent Capture by Aspiration (LASCA)
NASA Astrophysics Data System (ADS)
Brauer, Jonathan I.; Beech, Iwona B.; Sunner, Jan
2015-09-01
A novel interface for ambient, laser ablation-based mass spectrometric imaging (MSI) referred to as laser ablation and solvent capture by aspiration (LASCA) is presented and its performance demonstrated using selected, unaltered biological materials. LASCA employs a pulsed 2.94 μm laser beam for specimen ablation. Ablated materials in the laser plumes are collected on a hanging solvent droplet with electric field-enhanced trapping, followed by aspiration of droplets and remaining plume material in the form of a coarse aerosol into a collection capillary. The gas and liquid phases are subsequently separated in a 10 μL-volume separatory funnel, and the solution is analyzed with electrospray ionization in a high mass resolution Q-ToF mass spectrometer. The LASCA system separates the sampling and ionization steps in MSI and combines high efficiencies of laser plume sampling and of electrospray ionization (ESI) with high mass resolution MS. Up to 2000 different compounds are detected from a single ablation spot (pixel). Using the LASCA platform, rapid (6 s per pixel), high sensitivity, high mass-resolution ambient imaging of "as-received" biological material is achieved routinely and reproducibly.
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Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund
2017-05-31
Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.
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Optimal Background Estimators in Single-Molecule FRET Microscopy.
Preus, Søren; Hildebrandt, Lasse L; Birkedal, Victoria
2016-09-20
Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Förster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Application of the Attagene FACTORIAL™ assay to ...
Bioassays can be used to evaluate the integrated effects of complex mixtures from both known and unidentified contaminants present in environmental samples. However, such bio-monitoring approaches have typically focused only on one or a few pathways (e.g. estrogen receptor, androgen receptor) despite the fact that the chemicals in a mixture may exhibit a range of biological activities. High-throughput screening approaches that can rapidly assess samples for a broad diversity of biological activities offer a means to provide a more comprehensive characterization of complex mixtures. The Attagene FactorialTM platform is a high-throughput, cell based assay utilized by US EPA’s ToxCast Program, which provides high-content assessment of over 90 different gene regulatory pathways and all 48 human nuclear receptors (NRs). This assay has previously been used in a preliminary screening of surface water extracts from sites across the Great Lakes. In the current study, surface waters samples from 38 sites were collected, extracted, and screened through the Factorial assay as part of a USGS nationwide stream assessment. All samples were evaluated in a six point, 3-fold dilution series and analyzed using the ToxCast Data Pipeline (TCPL) to generate dose-response curves and corresponding half-maximal activity concentration (AC50) estimates. A total of 27 assay endpoints responded to extracts from one or more sites, with up to 14 assays active for a single extract. The four
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Walker, S. Hunter; Taylor, Amber D.; Muddiman, David C.
2013-01-01
The INLIGHT strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction due to isotopic overlap and a post-acquisition normalization of the data to account for both the systematic variability, a plot of the experimental H:L ratio vs. the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics. PMID:23860851
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Tips and traps in the 14C bio-AMS preparation laboratory
NASA Astrophysics Data System (ADS)
Buchholz, Bruce A.; Freeman, Stewart P. H. T.; Haack, Kurt W.; Vogel, John S.
2000-10-01
Maintaining a contamination free sample preparation lab for biological 14C AMS requires the same or more diligence as a radiocarbon dating prep lab. Isotope ratios of materials routinely range over 4-8 orders of magnitude in a single experiment, dosing solutions contain thousands of DPM and gels used to separate proteins possess 14C ratios of 1 amol 14C/mg C. Radiocarbon contamination is a legacy of earlier tracer work in most biological laboratories, even if they were never hot labs. Removable surface contamination can be found and monitored using swipes. Contamination can be found on any surface routinely touched: door knobs, light switches, drawer handles, water faucets. In general, all surfaces routinely touched need to be covered with paper, foil or plastic that can be changed frequently. Shared air supplies can also present problems by distributing hot aerosols throughout a building. Aerosols can be monitored for 14C content using graphitized coal or fullerene soot mixed with metal powder as an absorber. The monitors can be set out in work spaces for 1-2 weeks and measured by AMS with regular samples. Frequent air changes help minimize aerosol contamination in many cases. Cross-contamination of samples can be minimized by using disposable plastic or glassware in the prep lab, isolating samples from the air when possible and using positive displacement pipettors.
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Fessenden, S W; Hackmann, T J; Ross, D A; Foskolos, A; Van Amburgh, M E
2017-09-01
Microbial samples from 4 independent experiments in lactating dairy cattle were obtained and analyzed for nutrient composition, AA digestibility, and AA profile after multiple hydrolysis times ranging from 2 to 168 h. Similar bacterial and protozoal isolation techniques were used for all isolations. Omasal bacteria and protozoa samples were analyzed for AA digestibility using a new in vitro technique. Multiple time point hydrolysis and least squares nonlinear regression were used to determine the AA content of omasal bacteria and protozoa, and equivalency comparisons were made against single time point hydrolysis. Formalin was used in 1 experiment, which negatively affected AA digestibility and likely limited the complete release of AA during acid hydrolysis. The mean AA digestibility was 87.8 and 81.6% for non-formalin-treated bacteria and protozoa, respectively. Preservation of microbe samples in formalin likely decreased recovery of several individual AA. Results from the multiple time point hydrolysis indicated that Ile, Val, and Met hydrolyzed at a slower rate compared with other essential AA. Singe time point hydrolysis was found to be nonequivalent to multiple time point hydrolysis when considering biologically important changes in estimated microbial AA profiles. Several AA, including Met, Ile, and Val, were underpredicted using AA determination after a single 24-h hydrolysis. Models for predicting postruminal supply of AA might need to consider potential bias present in postruminal AA flow literature when AA determinations are performed after single time point hydrolysis and when using formalin as a preservative for microbial samples. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Morphomics: An integral part of systems biology of the human placenta.
Mayhew, T M
2015-04-01
The placenta is a transient organ the functioning of which has health consequences far beyond the embryo/fetus. Understanding the biology of any system (organ, organism, single cell, etc) requires a comprehensive and inclusive approach which embraces all the biomedical disciplines and 'omic' technologies and then integrates information obtained from all of them. Among the latest 'omics' is morphomics. The terms morphome and morphomics have been applied incoherently in biology and biomedicine but, recently, they have been given clear and widescale definitions. Morphomics is placed in the context of other 'omics' and its pertinent technologies and tools for sampling and quantitation are reviewed. Emphasis is accorded to the importance of random sampling principles in systems biology and the value of combining 3D quantification with alternative imaging techniques to advance knowledge and understanding of the human placental morphome. By analogy to other 'omes', the morphome is the totality of morphological features within a system and morphomics is the systematic study of those structures. Information about structure is required at multiple levels of resolution in order to understand better the processes by which a given system alters with time, experimental treatment or environmental insult. Therefore, morphomics research includes all imaging techniques at all levels of achievable resolution from gross anatomy and medical imaging, via optical and electron microscopy, to molecular characterisation. Quantification is an important element of all 'omics' studies and, because biological systems exist and operate in 3-dimensional (3D) space, precise descriptions of form, content and spatial relationships require the quantification of structure in 3D. These considerations are relevant to future study contributions to the Human Placenta Project. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Berger, Sebastian T; Ahmed, Saima; Muntel, Jan; Cuevas Polo, Nerea; Bachur, Richard; Kentsis, Alex; Steen, Judith; Steen, Hanno
2015-10-01
We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Microfluidic Platform for Parallel Single Cell Analysis for Diagnostic Applications.
Le Gac, Séverine
2017-01-01
Cell populations are heterogeneous: they can comprise different cell types or even cells at different stages of the cell cycle and/or of biological processes. Furthermore, molecular processes taking place in cells are stochastic in nature. Therefore, cellular analysis must be brought down to the single cell level to get useful insight into biological processes, and to access essential molecular information that would be lost when using a cell population analysis approach. Furthermore, to fully characterize a cell population, ideally, information both at the single cell level and on the whole cell population is required, which calls for analyzing each individual cell in a population in a parallel manner. This single cell level analysis approach is particularly important for diagnostic applications to unravel molecular perturbations at the onset of a disease, to identify biomarkers, and for personalized medicine, not only because of the heterogeneity of the cell sample, but also due to the availability of a reduced amount of cells, or even unique cells. This chapter presents a versatile platform meant for the parallel analysis of individual cells, with a particular focus on diagnostic applications and the analysis of cancer cells. We first describe one essential step of this parallel single cell analysis protocol, which is the trapping of individual cells in dedicated structures. Following this, we report different steps of a whole analytical process, including on-chip cell staining and imaging, cell membrane permeabilization and/or lysis using either chemical or physical means, and retrieval of the cell molecular content in dedicated channels for further analysis. This series of experiments illustrates the versatility of the herein-presented platform and its suitability for various analysis schemes and different analytical purposes.
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Kodandaramaiah, U; Weingartner, E; Janz, N; Dalén, L; Nylin, S
2011-10-01
Experimental work on Polygonia c-album, a temperate polyphagous butterfly species, has shown that Swedish, Belgian, Norwegian and Estonian females are generalists with respect to host-plant preference, whereas females from UK and Spain are specialized on Urticaceae. Female preference is known to have a strong genetic component. We test whether the specialist and generalist populations form respective genetic clusters using data from mitochondrial sequences and 10 microsatellite loci. Results do not support this hypothesis, suggesting that the specialist and generalist traits have evolved more than once independently. Mitochondrial DNA variation suggests a rapid expansion scenario, with a single widespread haplotype occurring in high frequency, whereas microsatellite data indicate strong differentiation of the Moroccan population. Based on a comparison of polymorphism in the mitochondrial data and sequences from a nuclear gene, we show that the diversity in the former is significantly less than that expected under neutral evolution. Furthermore, we found that almost all butterfly samples were infected with a single strain of Wolbachia, a maternally inherited bacterium. We reason that indirect selection on the mitochondrial genome mediated by a recent sweep of Wolbachia infection has depleted variability in the mitochondrial sequences. We also surmise that P. c-album could have expanded out of a single glacial refugium and colonized Morocco recently. © 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.
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Single-cell metabolomics: analytical and biological perspectives.
Zenobi, R
2013-12-06
There is currently much interest in broad molecular profiling of single cells; a cell's metabolome-its full complement of small-molecule metabolites-is a direct indicator of phenotypic diversity of single cells and a nearly immediate readout of how cells react to environmental influences. However, the metabolome is very difficult to measure at the single-cell level because of rapid metabolic dynamics, the structural diversity of the molecules, and the inability to amplify or tag small-molecule metabolites. Measurement techniques including mass spectrometry, capillary electrophoresis, and, to a lesser extent, optical spectroscopy and fluorescence detection have led to impressive advances in single-cell metabolomics. Even though none of these methodologies can currently measure the metabolome of a single cell completely, rapidly, and nondestructively, progress has been sufficient such that the field is witnessing a shift from feasibility studies to investigations that yield new biological insight. Particularly interesting fields of application are cancer biology, stem cell research, and monitoring of xenobiotics and drugs in tissue sections at the single-cell level.
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Single Cell Analysis: From Technology to Biology and Medicine.
Pan, Xinghua
2014-01-01
Single-cell analysis heralds a new era that allows "omics" analysis, notably genomics, transcriptomics, epigenomics and proteomics at the single-cell level. It enables the identification of the minor subpopulations that may play a critical role in a biological process of a population of cells, which conventionally are regarded as homogeneous. It provides an ultra-sensitive tool to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. It also facilitates the clinical investigation of patients when a very low quantity or a single cell is available for analysis, such as noninvasive prenatal diagnosis and cancer screening, and genetic evaluation for in vitro fertilization. Within a few short years, single-cell analysis, especially whole genomic sequencing and transcriptomic sequencing, is becoming robust and broadly accessible, although not yet a routine practice. Here, with single cell RNA-seq emphasized, an overview of the discipline, progresses, and prospects of single-cell analysis and its applications in biology and medicine are given with a series of logic and theoretical considerations.
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Stepanauskas, Ramunas
2018-02-06
DOE JGI's Tanja Woyke, chair of the Single Cells and Metagenomes session, delivers an introduction, followed by Bigelow Laboratory's Ramunas Stepanauskas on "Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.
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Preservation of Liquid Biological Samples
NASA Technical Reports Server (NTRS)
Putcha, Lakshmi (Inventor); Nimmagudda, Ramalingeshwara R. (Inventor)
2000-01-01
The present invention provides a method of preserving a liquid biological sample, comprising the step of: contacting said liquid biological sample with a preservative comprising, sodium benzoate in an amount of at least about 0.15% of the sample (weight/volume) and citric acid in an amount of at least about 0.025% of the sample (weight/volume).
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Zhang, L; Liu, X J
2016-06-03
With the rapid development of next-generation high-throughput sequencing technology, RNA-seq has become a standard and important technique for transcriptome analysis. For multi-sample RNA-seq data, the existing expression estimation methods usually deal with each single-RNA-seq sample, and ignore that the read distributions are consistent across multiple samples. In the current study, we propose a structured sparse regression method, SSRSeq, to estimate isoform expression using multi-sample RNA-seq data. SSRSeq uses a non-parameter model to capture the general tendency of non-uniformity read distribution for all genes across multiple samples. Additionally, our method adds a structured sparse regularization, which not only incorporates the sparse specificity between a gene and its corresponding isoform expression levels, but also reduces the effects of noisy reads, especially for lowly expressed genes and isoforms. Four real datasets were used to evaluate our method on isoform expression estimation. Compared with other popular methods, SSRSeq reduced the variance between multiple samples, and produced more accurate isoform expression estimations, and thus more meaningful biological interpretations.
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Concentration methods for high-resolution THz spectroscopy of nucleic-acid biomolecules and crystals
NASA Astrophysics Data System (ADS)
Brown, E. R.; Zhang, W.; Mendoza, E. A.; Kuznetsova, Y.; Brueck, S. R. J.; Rahman, M.; Norton, M. L.
2012-03-01
Biomolecules can exhibit low-lying vibrational modes in the THz region which are detectable in transmission given a strong molecular dipole moment and optical depth, and a spectrometer of adequate sensitivity. The nucleic acids are particularly interesting because of applications such as label-free gene assay, bio-agent detection, etc. However for nucleic acids, sample preparation and THz coupling are of paramount importance because of the strong absorption by liquid water and the small concentration of molecules present in physiological solutions. Concentration methods become necessary to make the THz vibrational modes detectable, either by concentrating the nucleic-acid sample itself in a small volume but large area, or by concentrating the THz radiation down to the volume of the sample. This paper summarizes one type of the first method: nanofluidic channel arrays for biological nucleic acids; and two types of the second method: (1) a circular-waveguide pinhole, and (2) a circular-waveguide, conical-horn coupling structure, both for DNA crystals. The first method has been demonstrated on a very short artificial nucleic acid [small-interfering (si) RNA (17-to-25 bp)] and a much longer, biological molecule [Lambda-phage DNA (48.5 kbp)]. The second method has been demonstrated on small (~100 micron) single crystals of DNA grown by the sitting-drop method.
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Sampson, Jason S.; Murray, Kermit K.; Muddiman, David C.
2013-01-01
We report the implementation of an infrared laser onto our previously reported matrix-assisted laser desorption electrospray ionization (MALDESI) source with ESI post-ionization yielding multiply charged peptides and proteins. Infrared (IR)-MALDESI is demonstrated for atmospheric pressure desorption and ionization of biological molecules ranging in molecular weight from 1.2 to 17 kDa. High resolving power, high mass accuracy single-acquisition Fourier transform ion cyclotron resonance (FT-ICR) mass spectra were generated from liquid-and solid-state peptide and protein samples by desorption with an infrared laser (2.94 µm) followed by ESI post-ionization. Intact and top-down analysis of equine myoglobin (17 kDa) desorbed from the solid state with ESI post-ionization demonstrates the sequencing capabilities using IR-MALDESI coupled to FT-ICR mass spectrometry. Carbohydrates and lipids were detected through direct analysis of milk and egg yolk using both UV- and IR-MALDESI with minimal sample preparation. Three of the four classes of biological macromolecules (proteins, carbohydrates, and lipids) have been ionized and detected using MALDESI with minimal sample preparation. Sequencing of O-linked glycans, cleaved from mucin using reductive β-elimination chemistry, is also demonstrated. PMID:19185512
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Malíková, M; Habrda, J
1976-03-01
Using two biological methods the chloramphenicol content in various tissues of 11 calves aged 3--18 days was determined after a single-dose therapeutical administration of the Czechoslovak preparation of Chronicin inj. in the quantity of 12 mg kg-1 body mass. Of four test strains, solely Micrococcus flavus (ATCC 10240) strain proved suitable. Chloramphenicol was quantitatively determined by reading from transformed standard curves. An evalua tion of the two approximately equally sensitive methods yielded the following results: three days after the injection all calves were positive, at 5 and 10 days, one of three calves in each group was negativ similarly to both calves slaughtered 15 days post injection. The residues appeared most frequently in the liver, at the site of injection, and in the fore and hind musculature. Maximum detectable concentrations were recorded in the liver. Culinary and technological treatment of positive meat and liver samples revealed that 15 minutes' boiling and 30 minutes' roasting destroyed the microbiologically active chloramphenicol residues in the samples. Neither deep freezing nor salting was sufficient for residue inactivation, although in some samples a decrease in biological chloramphenicol activity by circa 20--30% was recorded. a preslaughter withdrawal time of 15 days is recommended.
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Płonka, Joanna
2015-01-01
In recent years demands on the amount of information that can be obtained from the analysis of a single sample have increased. For time and economic reasons it is necessary to examine at the same time larger number of compounds, and compounds from different groups. This can best be seen in such areas as clinical analysis. In many diseases, the best results for patients are obtained when treatment fits the individual characteristics of the patient. Dosage monitoring is important at the beginning of therapy and in the full process of treatment. In the treatment of many diseases biogenic amines (dopamine, serotonin) and methylxanthines (theophylline, theobromine, caffeine) play an important role. They are used as drugs separately or in combination with others to support and strengthen the action of other drugs - for example, the combination of caffeine and paracetamol. Vitamin supplementation may be also an integral part of the treatment process. Specification of complete sample preparation parameters for extraction of the above compounds from biological matrices has been reviewed. Particular attention was given to the preparation stage and extraction methods. This review provides universal guidance on establishing a common procedures across laboratories to facilitate the preparation and analysis of all discussed compounds. Copyright © 2014 John Wiley & Sons, Ltd.
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Apparatus to collect, classify, concentrate, and characterize gas-borne particles
Rader, Daniel J.; Torczynski, John R.; Wally, Karl; Brockmann, John E.
2003-12-16
An aerosol lab-on-a-chip (ALOC) integrates one or more of a variety of particle collection, classification, concentration (enrichment), an characterization processes onto a single substrate or layered stack of such substrates. By mounting a UV laser diode laser light source on the substrate, or substrates tack, so that it is located down-stream of the sample inlet port and at right angle the sample particle stream, the UV light source can illuminate individual particles in the stream to induce a fluorescence response in those particles having a fluorescent signature such as biological particles, some of said particles. An illuminated particle having a fluorescent signal above a threshold signal would trigger a sorter module that would separate that particle from the particle stream.
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Genetic and educational assortative mating among US adults.
Domingue, Benjamin W; Fletcher, Jason; Conley, Dalton; Boardman, Jason D
2014-06-03
Understanding the social and biological mechanisms that lead to homogamy (similar individuals marrying one another) has been a long-standing issue across many fields of scientific inquiry. Using a nationally representative sample of non-Hispanic white US adults from the Health and Retirement Study and information from 1.7 million single-nucleotide polymorphisms, we compare genetic similarity among married couples to noncoupled pairs in the population. We provide evidence for genetic assortative mating in this population but the strength of this association is substantially smaller than the strength of educational assortative mating in the same sample. Furthermore, genetic similarity explains at most 10% of the assortative mating by education levels. Results are replicated using comparable data from the Framingham Heart Study.
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NASA Astrophysics Data System (ADS)
Grattieri, Matteo; Suvira, Milomir; Hasan, Kamrul; Minteer, Shelley D.
2017-07-01
The treatment of hypersaline wastewater (approximately 5% of the wastewater worldwide) cannot be performed by classical biological techniques. Herein the halotolerant extremophile bacteria obtained from the Great Salt Lake (Utah) were explored in single chamber microbial fuel cells with Pt-free cathodes for more than 18 days. The bacteria samples collected in two different locations of the lake (Stansbury Bay and Antelope Island) showed different electrochemical performances. The maximum achieved power output of 36 mW m-2 was from the microbial fuel cell based on the sample originated from Stansbury Bay, at a current density of 820 mA m-2. The performances throughout the long-term operation are discussed and a bioelectrochemical mechanism is proposed.
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Murphy, Gloria A [French Camp, CA
2010-09-07
A biological sample collector is adapted to a collect several biological samples in a plurality of filter wells. A biological sample collector may comprise a manifold plate for mounting a filter plate thereon, the filter plate having a plurality of filter wells therein; a hollow slider for engaging and positioning a tube that slides therethrough; and a slide case within which the hollow slider travels to allow the tube to be aligned with a selected filter well of the plurality of filter wells, wherein when the tube is aligned with the selected filter well, the tube is pushed through the hollow slider and into the selected filter well to sealingly engage the selected filter well and to allow the tube to deposit a biological sample onto a filter in the bottom of the selected filter well. The biological sample collector may be portable.
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Brabencová, Sylva; Ihnatová, Ivana; Potěšil, David; Fojtová, Miloslava; Fajkus, Jiří; Zdráhal, Zbyněk; Lochmanová, Gabriela
2017-01-01
Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles. PMID:29270186
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Brabencová, Sylva; Ihnatová, Ivana; Potěšil, David; Fojtová, Miloslava; Fajkus, Jiří; Zdráhal, Zbyněk; Lochmanová, Gabriela
2017-01-01
Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.
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The Difference a Single Atom Can Make: Synthesis and Design at the Chemistry–Biology Interface
2017-01-01
A Perspective of work in our laboratory on the examination of biologically active compounds, especially natural products, is presented. In the context of individual programs and along with a summary of our work, selected cases are presented that illustrate the impact single atom changes can have on the biological properties of the compounds. The examples were chosen to highlight single heavy atom changes that improve activity, rather than those that involve informative alterations that reduce or abolish activity. The examples were also chosen to illustrate that the impact of such single-atom changes can originate from steric, electronic, conformational, or H-bonding effects, from changes in functional reactivity, from fundamental intermolecular interactions with a biological target, from introduction of a new or altered functionalization site, or from features as simple as improvements in stability or physical properties. Nearly all the examples highlighted represent not only unusual instances of productive deep-seated natural product modifications and were introduced through total synthesis but are also remarkable in that they are derived from only a single heavy atom change in the structure. PMID:28945374
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Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy
Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent
2011-01-01
We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327
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Counting Unfolding Events in Stretched Helices with Induced Oscillation by Optical Tweezers
NASA Astrophysics Data System (ADS)
Bacabac, Rommel Gaud; Otadoy, Roland
Correlation measures based on embedded probe fluctuations, single or paired, are now widely used for characterizing the viscoelastic properties of biological samples. However, more robust applications using this technique are still lacking. Considering that the study of living matter routinely demonstrates new and complex phenomena, mathematical and experimental tools for analysis have to catch up in order to arrive at newer insights. Therefore, we derive ways of probing non-equilibrium events in helical biopolymers provided by stretching beyond thermal forces. We generalize, for the first time, calculations for winding turn probabilities to account for unfolding events in single fibrous biopolymers and globular proteins under tensile stretching using twin optical traps. The approach is based on approximating the ensuing probe fluctuations as originating from a damped harmonic oscillator under oscillatory forcing.
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Dual function microscope for quantitative DIC and birefringence imaging
NASA Astrophysics Data System (ADS)
Li, Chengshuai; Zhu, Yizheng
2016-03-01
A spectral multiplexing interferometry (SXI) method is presented for integrated birefringence and phase gradient measurement on label-free biological specimens. With SXI, the retardation and orientation of sample birefringence are simultaneously encoded onto two separate spectral carrier waves, generated by a crystal retarder oriented at a specific angle. Thus sufficient information for birefringence determination can be obtained from a single interference spectrum, eliminating the need for multiple acquisitions with mechanical rotation or electrical modulation. In addition, with the insertion of a Nomarski prism, the setup can then acquire quantitative differential interference contrast images. Red blood cells infected by malaria parasites are imaged for birefringence retardation as well as phase gradient. The results demonstrate that the SXI approach can achieve both quantitative phase imaging and birefringence imaging with a single, high-sensitivity system.
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The system spatial-frequency filtering of birefringence images of human blood layers
NASA Astrophysics Data System (ADS)
Ushenko, A. G.; Boychuk, T. M.; Mincer, O. P.; Angelsky, P. O.; Bodnar, N. B.; Oleinichenko, B. P.; Bizer, L. I.
2013-09-01
Among various opticophysical methods [1 - 3] of diagnosing the structure and properties of the optical anisotropic component of various biological objects a specific trend has been singled out - multidimensional laser polarimetry of microscopic images of the biological tissues with the following statistic, correlative and fractal analysis of the coordinate distributions of the azimuths and ellipticity of polarization in approximating of linear birefringence polycrystalline protein networks [4 - 10]. At the same time, in most cases, experimental obtaining of tissue sample is a traumatic biopsy operation. In addition, the mechanisms of transformation of the state of polarization of laser radiation by means of the opticoanisotropic biological structures are more varied (optical dichroism, circular birefringence). Hereat, real polycrystalline networks can be formed by different types, both in size and optical properties of biological crystals. Finally, much more accessible for an experimental investigation are biological fluids such as blood, bile, urine, and others. Thus, further progress of laser polarimetry can be associated with the development of new methods of analysis and processing (selection) of polarization- heterogeneous images of biological tissues and fluids, taking into account a wider set of mechanisms anisotropic mechanisms. Our research is aimed at developing experimental method of the Fourier polarimetry and a spatialfrequency selection for distributions of the azimuth and the ellipticity polarization of blood plasma laser images with a view of diagnosing prostate cancer.
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Differential principal component analysis of ChIP-seq.
Ji, Hongkai; Li, Xia; Wang, Qian-fei; Ning, Yang
2013-04-23
We propose differential principal component analysis (dPCA) for analyzing multiple ChIP-sequencing datasets to identify differential protein-DNA interactions between two biological conditions. dPCA integrates unsupervised pattern discovery, dimension reduction, and statistical inference into a single framework. It uses a small number of principal components to summarize concisely the major multiprotein synergistic differential patterns between the two conditions. For each pattern, it detects and prioritizes differential genomic loci by comparing the between-condition differences with the within-condition variation among replicate samples. dPCA provides a unique tool for efficiently analyzing large amounts of ChIP-sequencing data to study dynamic changes of gene regulation across different biological conditions. We demonstrate this approach through analyses of differential chromatin patterns at transcription factor binding sites and promoters as well as allele-specific protein-DNA interactions.
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Surface-enhanced Raman detection of CW agents in water using gold sol gel substrates
NASA Astrophysics Data System (ADS)
Premasiri, W. Ranjith; Clarke, Richard H.; Womble, M. Edward
2002-02-01
The development of a water analysis system capable of detecting both inanimate trace chemical contaminants and viable microbial contaminants has long been a project of interest to our group. The capability of detecting both chemical and biological agent sources in a single device configuration would clearly add to the value of such a product. In the present work, we describe results with chemical warfare agents from our efforts to produce a Raman system for the detection of both chemical and biological warfare agents in water. We utilize laser Raman light scattering and employ Surface Enhanced Raman Spectroscopy (SERS)on solid state gold sol-gel detectors combined with fiber optic collection of the enhanced light signal in the sampling system to augment the normally low intensity Raman Scattering signal from trace materials.
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Myzithras, Maria; Li, Hua; Bigwarfe, Tammy; Waltz, Erica; Gupta, Priyanka; Low, Sarah; Hayes, David B; MacDonnell, Scott; Ahlberg, Jennifer; Franti, Michael; Roberts, Simon
2016-03-01
Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.
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Rosen, David B.; Minden, Mark D.; Kornblau, Steven M.; Cohen, Aileen; Gayko, Urte; Putta, Santosh; Woronicz, John; Evensen, Erik; Fantl, Wendy J.; Cesano, Alessandra
2010-01-01
Background Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative. Methodology/Principal Findings Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management. Conclusions/Significance These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations. PMID:21048955
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A novel conformation of gel grown biologically active cadmium nicotinate
NASA Astrophysics Data System (ADS)
Nair, Lekshmi P.; Bijini, B. R.; Divya, R.; Nair, Prabitha B.; Eapen, S. M.; Dileep Kumar, B. S.; Nishanth Kumar, S.; Nair, C. M. K.; Deepa, M.; Rajendra Babu, K.
2017-11-01
The elimination of toxic heavy metals by the formation of stable co-ordination compounds with biologically active ligands is applicable in drug designing. A new crystalline complex of cadmium with nicotinic acid is grown at ambient temperature using the single gel diffusion method in which the crystal structure is different from those already reported. Single crystal x-ray diffraction reveals the identity of crystal structure belonging to monoclinic system, P21/c space group with cell dimensions a = 17.220 (2) Å, b = 10.2480 (2) Å, c = 7.229(9) Å, β = 91.829(4)°. Powder x-ray diffraction analysis confirmed the crystallinity of the sample. The unidentate mode of co-ordination between the metal atom and the carboxylate group is supported by the Fourier Transform Infra Red spectral data. Thermal analysis ensures the thermal stability of the complex. Kinetic and thermodynamic parameters are also calculated. The stoichiometry of the complex is confirmed by the elemental analysis. The UV-visible spectral analysis shows the wide transparency window of the complex in the visible region. The band gap of the complex is found to be 3.92 eV. The complex shows excellent antibacterial and antifungal activity.
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THE RABIT: A RAPID AUTOMATED BIODOSIMETRY TOOL FOR RADIOLOGICAL TRIAGE
Garty, Guy; Chen, Youhua; Salerno, Alessio; Turner, Helen; Zhang, Jian; Lyulko, Oleksandra; Bertucci, Antonella; Xu, Yanping; Wang, Hongliang; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y. Lawrence; Amundson, Sally A.; Brenner, David J.
2010-01-01
In response to the recognized need for high throughput biodosimetry methods for use after large scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. We describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or γ-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine, to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in-situ analysis in multi-well plates, implementation of robotic plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here we describe the development of each of the individual modules of the RABIT system, and show preliminary data from key modules. Ongoing is system integration, followed by calibration and validation. PMID:20065685
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NASA Astrophysics Data System (ADS)
Augustin-Bauditz, Stefanie; Wex, Heike; Denjean, Cyrielle; Hartmann, Susan; Schneider, Johannes; Schmidt, Susann; Ebert, Martin; Stratmann, Frank
2016-05-01
Biological particles such as bacteria, fungal spores or pollen are known to be efficient ice nucleating particles. Their ability to nucleate ice is due to ice nucleation active macromolecules (INMs). It has been suggested that these INMs maintain their nucleating ability even when they are separated from their original carriers. This opens the possibility of an accumulation of such INMs in soils, resulting in an internal mixture of mineral dust and INMs. If particles from such soils which contain biological INMs are then dispersed into the atmosphere due to wind erosion or agricultural processes, they could induce ice nucleation at temperatures typical for biological substances, i.e., above -20 up to almost 0 °C, while they might be characterized as mineral dust particles due to a possibly low content of biological material. We conducted a study within the research unit INUIT (Ice Nucleation research UnIT), where we investigated the ice nucleation behavior of mineral dust particles internally mixed with INM. Specifically, we mixed a pure mineral dust sample (illite-NX) with ice active biological material (birch pollen washing water) and quantified the immersion freezing behavior of the resulting particles utilizing the Leipzig Aerosol Cloud Interaction Simulator (LACIS). A very important topic concerning the investigations presented here as well as for atmospheric application is the characterization of the mixing state of aerosol particles. In the present study we used different methods like single-particle aerosol mass spectrometry, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray analysis (EDX), and a Volatility-Hygroscopicity Tandem Differential Mobility Analyser (VH-TDMA) to investigate the mixing state of our generated aerosol. Not all applied methods performed similarly well in detecting small amounts of biological material on the mineral dust particles. Measuring the hygroscopicity/volatility of the mixed particles with the VH-TDMA was the most sensitive method. We found that internally mixed particles, containing ice active biological material, follow the ice nucleation behavior observed for the pure biological particles. We verified this by modeling the freezing behavior of the mixed particles with the Soccerball model (SBM). It can be concluded that a single INM located on a mineral dust particle determines the freezing behavior of that particle with the result that freezing occurs at temperatures at which pure mineral dust particles are not yet ice active.
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Levine, Keith E; Redmon, Jennifer Hoponick; Elledge, Myles F; Wanigasuriya, Kamani P; Smith, Kristin; Munoz, Breda; Waduge, Vajira A; Periris-John, Roshini J; Sathiakumar, Nalini; Harrington, James M; Womack, Donna S; Wickremasinghe, Rajitha
2016-10-01
The emergence of a new form of chronic kidney disease of unknown etiology (CKDu) in Sri Lanka's North Central Province (NCP) has become a catastrophic health crisis. CKDu is characterized as slowly progressing, irreversible, and asymptomatic until late stages and, importantly, not attributed to diabetes, hypertension, or other known risk factors. It is postulated that the etiology of CKDu is multifactorial, involving genetic predisposition, nutritional and dehydration status, exposure to one or more environmental nephrotoxins, and lifestyle factors. The objective of this limited geochemical laboratory analysis was to determine the concentration of a suite of heavy metals and trace element nutrients in biological samples (human whole blood and hair) and environmental samples (drinking water, rice, soil, and freshwater fish) collected from two towns within the endemic NCP region in 2012 and 2013. This broad panel, metallomics/mineralomics approach was used to shed light on potential geochemical risk factors associated with CKDu. Based on prior literature documentation of potential nephrotoxins that may play a role in the genesis and progression of CKDu, heavy metals and fluoride were selected for analysis. The geochemical concentrations in biological and environmental media areas were quantified. Basic statistical measurements were subsequently used to compare media against applicable benchmark values, such as US soil screening levels. Cadmium, lead, and mercury were detected at concentrations exceeding US reference values in many of the biological samples, suggesting that study participants are subjected to chronic, low-level exposure to these elements. Within the limited number of environmental media samples, arsenic was determined to exceed initial risk screening and background concentration values in soil, while data collected from drinking water samples reflected the unique hydrogeochemistry of the region, including the prevalence of hard or very hard water, and fluoride, iron, manganese, sodium, and lead exceeding applicable drinking water standards in some instances. Current literature suggests that the etiology of CKDu is likely multifactorial, with no single biological or hydrogeochemical parameter directly related to disease genesis and progression. This preliminary screening identified that specific constituents may be present above levels of concern, but does not compare results against specific kidney toxicity values or cumulative risk related to a multifactorial disease process. The data collected from this limited investigation are intended to be used in the subsequent study design of a comprehensive and multifactorial etiological study of CKDu risk factors that includes sample collection, individual surveys, and laboratory analyses to more fully evaluate the potential environmental, behavioral, genetic, and lifestyle risk factors associated with CKDu.
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Biogenic magnetite as a primary remanence carrier in limestone deposits
NASA Astrophysics Data System (ADS)
Chang, Shih-Bin R.; Kirschvink, Joseph L.; Stolz, John F.
1987-06-01
Studies on the microbial communities and magnetic phases of samples collected from carbonate oozes at Sugarloaf Key, FL, U.S.A. and calcareous laminated sediments from Laguna Figueroa, Baja California, Mexico have revealed the existence of magnetotactic bacteria and ultrafine-grained single domain magnetite in both environments. Magnetotactic bacteria were identified by light and electron microscopy. The single domain magnetite was detected by coercivity spectra analysis with a SQUID magnetometer and examined under the transmission electron microscope. The similarity, in terms of size and shape, between the single domain magnetite found in these sediments and the magnetite observed in the bacterial magnetosome from enriched cultures indicates the ultrafine-grained magnetite in these two marine environments was biologically formed. These results, combined with the common occurrences of ultrafine-grained magnetite in limestone deposits detected rock magnetically, suggest biogenic magnetite may be present and contribute to the magnetic remanence in these rocks. Several Cambrian limestone samples, separately collected from Siberia, China, and Kazakhstan, were examined for the presence of bacterial magnetite. Samples from the Lower Cambrian Sinskian Formation at Siberia Platform were found to contain both a large amount of apparently bacterial magnetite particles and a very stable primary magnetic component. Post-Cambrian diagenesis does not seem to affect the microgranulometry of these apparently bacterial magnetite crystals or the magnetic remanence carried by them. Assessing the potential role of biogenic magnetite as a primary remanence carrier in other Phanerozoic limestone deposits ought to be further pursued.
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Tu, Bin; Masaberg, Carly; Hou, Lihua; Behm, Daniel; Brescia, Peter; Cha, Nuri; Kariyawasam, Kanthi; Lee, Jar How; Nong, Thoa; Sells, John; Tausch, Paul; Yang, Ruyan; Ng, Jennifer; Hurley, Carolyn Katovich
2017-02-01
Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons. Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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STBase: One Million Species Trees for Comparative Biology
McMahon, Michelle M.; Deepak, Akshay; Fernández-Baca, David; Boss, Darren; Sanderson, Michael J.
2015-01-01
Comprehensively sampled phylogenetic trees provide the most compelling foundations for strong inferences in comparative evolutionary biology. Mismatches are common, however, between the taxa for which comparative data are available and the taxa sampled by published phylogenetic analyses. Moreover, many published phylogenies are gene trees, which cannot always be adapted immediately for species level comparisons because of discordance, gene duplication, and other confounding biological processes. A new database, STBase, lets comparative biologists quickly retrieve species level phylogenetic hypotheses in response to a query list of species names. The database consists of 1 million single- and multi-locus data sets, each with a confidence set of 1000 putative species trees, computed from GenBank sequence data for 413,000 eukaryotic taxa. Two bodies of theoretical work are leveraged to aid in the assembly of multi-locus concatenated data sets for species tree construction. First, multiply labeled gene trees are pruned to conflict-free singly-labeled species-level trees that can be combined between loci. Second, impacts of missing data in multi-locus data sets are ameliorated by assembling only decisive data sets. Data sets overlapping with the user’s query are ranked using a scheme that depends on user-provided weights for tree quality and for taxonomic overlap of the tree with the query. Retrieval times are independent of the size of the database, typically a few seconds. Tree quality is assessed by a real-time evaluation of bootstrap support on just the overlapping subtree. Associated sequence alignments, tree files and metadata can be downloaded for subsequent analysis. STBase provides a tool for comparative biologists interested in exploiting the most relevant sequence data available for the taxa of interest. It may also serve as a prototype for future species tree oriented databases and as a resource for assembly of larger species phylogenies from precomputed trees. PMID:25679219
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Ward, Joey; Strawbridge, Rona J; Bailey, Mark E S; Graham, Nicholas; Ferguson, Amy; Lyall, Donald M; Cullen, Breda; Pidgeon, Laura M; Cavanagh, Jonathan; Mackay, Daniel F; Pell, Jill P; O'Donovan, Michael; Escott-Price, Valentina; Smith, Daniel J
2017-11-30
Mood instability is a core clinical feature of affective and psychotic disorders. In keeping with the Research Domain Criteria approach, it may be a useful construct for identifying biology that cuts across psychiatric categories. We aimed to investigate the biological validity of a simple measure of mood instability and evaluate its genetic relationship with several psychiatric disorders, including major depressive disorder (MDD), bipolar disorder (BD), schizophrenia, attention deficit hyperactivity disorder (ADHD), anxiety disorder and post-traumatic stress disorder (PTSD). We conducted a genome-wide association study (GWAS) of mood instability in 53,525 cases and 60,443 controls from UK Biobank, identifying four independently associated loci (on chromosomes 8, 9, 14 and 18), and a common single-nucleotide polymorphism (SNP)-based heritability estimate of ~8%. We found a strong genetic correlation between mood instability and MDD (r g = 0.60, SE = 0.07, p = 8.95 × 10 -17 ) and a small but significant genetic correlation with both schizophrenia (r g = 0.11, SE = 0.04, p = 0.01) and anxiety disorders (r g = 0.28, SE = 0.14, p = 0.04), although no genetic correlation with BD, ADHD or PTSD was observed. Several genes at the associated loci may have a role in mood instability, including the DCC netrin 1 receptor (DCC) gene, eukaryotic translation initiation factor 2B subunit beta (eIF2B2), placental growth factor (PGF) and protein tyrosine phosphatase, receptor type D (PTPRD). Strengths of this study include the very large sample size, but our measure of mood instability may be limited by the use of a single question. Overall, this work suggests a polygenic basis for mood instability. This simple measure can be obtained in very large samples; our findings suggest that doing so may offer the opportunity to illuminate the fundamental biology of mood regulation.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Steele, Paul Thomas
2004-09-01
Better devices are needed for the detection of aerosolized biological warfare agents. Advances in the ongoing development of one such device, the BioAerosol Mass Spectrometry (BAMS) system, are described here in detail. The system samples individual, micrometer-sized particles directly from the air and analyzes them in real-time without sample preparation or use of reagents. At the core of the BAMS system is a dual-polarity, single-particle mass spectrometer with a laser based desorption and ionization (DI) system. The mass spectra produced by early proof-of-concept instruments were highly variable and contained limited information to differentiate certain types of similar biological particles. Themore » investigation of this variability and subsequent changes to the DI laser system are described. The modifications have reduced the observed variability and thereby increased the usable information content in the spectra. These improvements would have little value without software to analyze and identify the mass spectra. Important improvements have been made to the algorithms that initially processed and analyzed the data. Single particles can be identified with an impressive level of accuracy, but to obtain significant reductions in the overall false alarm rate of the BAMS instrument, alarm decisions must be made dynamically on the basis of multiple analyzed particles. A statistical model has been developed to make these decisions and the resulting performance of a hypothetical BAMS system is quantitatively predicted. The predictions indicate that a BAMS system, with reasonably attainable characteristics, can operate with a very low false alarm rate (orders of magnitude lower than some currently fielded biodetectors) while still being sensitive to small concentrations of biological particles in a large range of environments. Proof-of-concept instruments, incorporating some of the modifications described here, have already performed well in independent testing.« less
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Hemasa, Ayman L.; Maher, William A.; Ghanem, Ashraf
2017-01-01
Carbon nanotubes (CNTs) possess unique mechanical, physical, electrical and absorbability properties coupled with their nanometer dimensional scale that renders them extremely valuable for applications in many fields including nanotechnology and chromatographic separation. The aim of this review is to provide an updated overview about the applications of CNTs in chiral and achiral separations of pharmaceuticals, biologics and chemicals. Chiral single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) have been directly applied for the enantioseparation of pharmaceuticals and biologicals by using them as stationary or pseudostationary phases in chromatographic separation techniques such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and gas chromatography (GC). Achiral MWCNTs have been used for achiral separations as efficient sorbent objects in solid-phase extraction techniques of biochemicals and drugs. Achiral SWCNTs have been applied in achiral separation of biological samples. Achiral SWCNTs and MWCNTs have been also successfully used to separate achiral mixtures of pharmaceuticals and chemicals. Collectively, functionalized CNTs have been indirectly applied in separation science by enhancing the enantioseparation of different chiral selectors whereas non-functionalized CNTs have shown efficient capabilities for chiral separations by using techniques such as encapsulation or immobilization in polymer monolithic columns. PMID:28718832
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Wang, Han; Chen, Beibei; He, Man; Hu, Bin
2017-05-02
Single cell analysis is a significant research field in recent years reflecting the heterogeneity of cells in a biological system. In this work, a facile droplet chip was fabricated and online combined with time-resolved inductively coupled plasma mass spectrometry (ICPMS) via a microflow nebulizer for the determination of zinc in single HepG2 cells. On the focusing geometric designed PDMS microfluidic chip, the aqueous cell suspension was ejected and divided by hexanol to generate droplets. The droplets encapsulated single cells remain intact during the transportation into ICP for subsequent detection. Under the optimized conditions, the frequency of droplet generation is 3-6 × 10 6 min -1 , and the injected cell number is 2500 min -1 , which can ensure the single cell encapsulation. ZnO nanoparticles (NPs) were used for the quantification of zinc in single cells, and the accuracy was validated by conventional acid digestion-ICPMS method. The ZnO NPs incubated HepG2 cells were analyzed as model samples, and the results exhibit the heterogeneity of HepG2 cells in the uptake/adsorption of ZnO NPs. The developed online droplet-chip-ICPMS analysis system achieves stable single cell encapsulation and has high throughput for single cell analysis. It has the potential in monitoring the content as well as distribution of trace elements/NPs at the single cell level.
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Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.
Song, Linan; Ahn, Soohyoun; Walt, David R
2006-02-15
We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.
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Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.
Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi
2017-12-21
High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.
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Grey matter networks in people at increased familial risk for schizophrenia.
Tijms, Betty M; Sprooten, Emma; Job, Dominic; Johnstone, Eve C; Owens, David G C; Willshaw, David; Seriès, Peggy; Lawrie, Stephen M
2015-10-01
Grey matter brain networks are disrupted in schizophrenia, but it is still unclear at which point during the development of the illness these disruptions arise and whether these can be associated with behavioural predictors of schizophrenia. We investigated if single-subject grey matter networks were disrupted in a sample of people at familial risk of schizophrenia. Single-subject grey matter networks were extracted from structural MRI scans of 144 high risk subjects, 32 recent-onset patients and 36 healthy controls. The following network properties were calculated: size, connectivity density, degree, path length, clustering coefficient, betweenness centrality and small world properties. People at risk of schizophrenia showed decreased path length and clustering in mostly prefrontal and temporal areas. Within the high risk sample, the path length of the posterior cingulate cortex and the betweenness centrality of the left inferior frontal operculum explained 81% of the variance in schizotypal cognitions, which was previously shown to be the strongest behavioural predictor of schizophrenia in the study. In contrast, local grey matter volume measurements explained 48% of variance in schizotypy. The present results suggest that single-subject grey matter networks can quantify behaviourally relevant biological alterations in people at increased risk for schizophrenia before disease onset. Copyright © 2015 Elsevier B.V. All rights reserved.
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Pleil, Joachim D; Angrish, Michelle M; Madden, Michael C
2015-12-11
Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-α, IL-1β and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r(2) > 0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg ml(-1). All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ~45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml(-1)). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels.
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Detectors for single-molecule fluorescence imaging and spectroscopy
MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.
2010-01-01
Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633
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Bulbul, Gonca; Chaves, Gepoliano; Olivier, Joseph; Ozel, Rifat Emrah; Pourmand, Nader
2018-06-06
Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology.
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Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.
Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido
2015-02-01
Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.
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A Problem-Sorting Task Detects Changes in Undergraduate Biological Expertise over a Single Semester.
Hoskinson, Anne-Marie; Maher, Jessica Middlemis; Bekkering, Cody; Ebert-May, Diane
2017-01-01
Calls for undergraduate biology reform share similar goals: to produce people who can organize, use, connect, and communicate about biological knowledge. Achieving these goals requires students to gain disciplinary expertise. Experts organize, access, and apply disciplinary knowledge differently than novices, and expertise is measurable. By asking introductory biology students to sort biological problems, we investigated whether they changed how they organized and linked biological ideas over one semester of introductory biology. We administered the Biology Card Sorting Task to 751 students enrolled in their first or second introductory biology course focusing on either cellular-molecular or organismal-population topics, under structured or unstructured sorting conditions. Students used a combination of superficial, deep, and yet-uncharacterized ways of organizing and connecting biological knowledge. In some cases, this translated to more expert-like ways of organizing knowledge over a single semester, best predicted by whether students were enrolled in their first or second semester of biology and by the sorting condition completed. In addition to illuminating differences between novices and experts, our results show that card sorting is a robust way of detecting changes in novices' biological expertise-even in heterogeneous populations of novice biology students over the time span of a single semester. © 2017 A.-M. Hoskinson et al. CBE—Life Sciences Education © 2017 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License(http://creativecommons.org/licenses/by-nc-sa/3.0).
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Distribution of Amino Acids in Lunar Regolith
NASA Technical Reports Server (NTRS)
Elsila, J. E.; Callahan, M. P.; Glavin, D. P.; Dworkin, J. P.; Noble, S. K.; Gibson, E. K., Jr.
2014-01-01
One of the most eagerly studied questions upon initial return of lunar samples was whether significant amounts of organic compounds, including amino acids, were present. Analyses during the 1970s produced only tentative and inconclusive identifications of indigenous amino acids. Those analyses were hampered by analytical difficulties including relative insensitivity to certain compounds, the inability to separate chiral enantiomers, and the lack of compound-specific isotopic measurements, which made it impossible to determine whether the detected amino acids were indigenous to the lunar samples or the results of contamination. Numerous advances have been made in instrumentation and methodology for amino acid characterization in extraterrestrial samples in the intervening years, yet the origin of amino acids in lunar regolith samples has been revisited only once for a single lunar sample, (3) and remains unclear. Here, we present initial data from the analyses of amino acid abundances in 12 lunar regolith samples. We discuss these abundances in the context of four potential amino acid sources: (1) terrestrial biological contamination; (2) contamination from lunar module (LM) exhaust; (3) derivation from solar windimplanted precursors; and (4) exogenous delivery from meteorites.
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NASA Astrophysics Data System (ADS)
Poola, Praveen Kumar; John, Renu
2017-10-01
We report the results of characterization of red blood cell (RBC) structure and its dynamics with nanometric sensitivity using transport of intensity equation microscopy (TIEM). Conventional transport of intensity technique requires three intensity images and hence is not suitable for studying real-time dynamics of live biological samples. However, assuming the sample to be homogeneous, phase retrieval using transport of intensity equation has been demonstrated with single defocused measurement with x-rays. We adopt this technique for quantitative phase light microscopy of homogenous cells like RBCs. The main merits of this technique are its simplicity, cost-effectiveness, and ease of implementation on a conventional microscope. The phase information can be easily merged with regular bright-field and fluorescence images to provide multidimensional (three-dimensional spatial and temporal) information without any extra complexity in the setup. The phase measurement from the TIEM has been characterized using polymeric microbeads and the noise stability of the system has been analyzed. We explore the structure and real-time dynamics of RBCs and the subdomain membrane fluctuations using this technique.
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Aerobiology: Experimental Considerations, Observations, and Future Tools
Haddrell, Allen E.
2017-01-01
ABSTRACT Understanding airborne survival and decay of microorganisms is important for a range of public health and biodefense applications, including epidemiological and risk analysis modeling. Techniques for experimental aerosol generation, retention in the aerosol phase, and sampling require careful consideration and understanding so that they are representative of the conditions the bioaerosol would experience in the environment. This review explores the current understanding of atmospheric transport in relation to advances and limitations of aerosol generation, maintenance in the aerosol phase, and sampling techniques. Potential tools for the future are examined at the interface between atmospheric chemistry, aerosol physics, and molecular microbiology where the heterogeneity and variability of aerosols can be explored at the single-droplet and single-microorganism levels within a bioaerosol. The review highlights the importance of method comparison and validation in bioaerosol research and the benefits that the application of novel techniques could bring to increasing the understanding of aerobiological phenomena in diverse research fields, particularly during the progression of atmospheric transport, where complex interdependent physicochemical and biological processes occur within bioaerosol particles. PMID:28667111
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On the shock response of pisum sativum and lepidium sativum
NASA Astrophysics Data System (ADS)
Leighs, James Allen; Hazell, Paul; Appleby-Thomas, Gareth James
2012-03-01
The high strain-rate response of biological and organic structures is of interest to numerous fields ranging from the food industry to astrobiology. Consequently, knowledge of the damage mechanisms within, and the viability of shocked organic material are of significant importance. In this study, a single-stage gasgun has been employed to subject samples of Pisum sativum (common pea) and Lepidium sativum (curled cress) to planar shock loading. Impact pressures of up to ~11.5 GPa and ~0.5 GPa for pea and cress seed samples respectively have been reached. The development of the experimental approach is discussed and presented alongside results from modelled gauge traces showing the sample loading history. Viability of the shock-loaded pea and cress seeds was investigated via attempts at germination, which were unsuccessful with pea seeds but successful in all tests performed on cress seeds. This work suggests that organic structures could survive shockwaves that may be encountered during asteroid collisions.
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Cuffney, Thomas F.; Gurtz, Martin E.; Meador, Michael R.
1993-01-01
Benthic invertebrate communities are evaluated as part of the ecological survey component of the U.S. Geological Survey's National Water-Quality Assessment Program. These biological data are collected along with physical and chemical data to assess water-quality conditions and to develop an understanding of the factors that affect water-quality conditions locally, regionally, and nationally. The objectives of benthic invertebrate community characterizations are to (1) develop for each site a list of tax a within the associated stream reach and (2) determine the structure of benthic invertebrate communities within selected habitats of that reach. A nationally consistent approach is used to achieve these objectives. This approach provides guidance on site, reach, and habitat selection and methods and equipment for qualitative multihabitat sampling and semi-quantitative single habitat sampling. Appropriate quality-assurance and quality-control guidelines are used to maximize the ability to analyze data within and among study units.
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NASA Astrophysics Data System (ADS)
Hsueh, Carlin
Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an interaction that can provide scientists with a better understanding of debilitating neurological diseases, such as Alzheimer's and Down Syndrome at the molecular level. These observations provide extraordinary access to the subtle signs and behavior indicating early onset of disease in cells and tissues and to the dynamics of disease development and treatment.
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Atomic force microscopy imaging of macromolecular complexes.
Santos, Sergio; Billingsley, Daniel; Thomson, Neil
2013-01-01
This chapter reviews amplitude modulation (AM) AFM in air and its applications to high-resolution imaging and interpretation of macromolecular complexes. We discuss single DNA molecular imaging and DNA-protein interactions, such as those with topoisomerases and RNA polymerase. We show how relative humidity can have a major influence on resolution and contrast and how it can also affect conformational switching of supercoiled DNA. Four regimes of AFM tip-sample interaction in air are defined and described, and relate to water perturbation and/or intermittent mechanical contact of the tip with either the molecular sample or the surface. Precise control and understanding of the AFM operational parameters is shown to allow the user to switch between these different regimes: an interpretation of the origins of topographical contrast is given for each regime. Perpetual water contact is shown to lead to a high-resolution mode of operation, which we term SASS (small amplitude small set-point) imaging, and which maximizes resolution while greatly decreasing tip and sample wear and any noise due to perturbation of the surface water. Thus, this chapter provides sufficient information to reliably control the AFM in the AM AFM mode of operation in order to image both heterogeneous samples and single macromolecules including complexes, with high resolution and with reproducibility. A brief introduction to AFM, its versatility and applications to biology is also given while providing references to key work and general reviews in the field.
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Mark T. Banik; Harold H. Burdsall
1998-01-01
Ten single-spore isolates each of Armillaria sinapina, A. cepistipes, and North American biological species (NABS)X and XI were paired in all combinations. A second set of ten single-spore isolates of each species was likewise paired. Each pairing was duplicated for a total of 3280 pairs. Using the standard morphological criteria (e.g., fluffy, crustose) to assess the...
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Single molecules can operate as primitive biological sensors, switches and oscillators.
Hernansaiz-Ballesteros, Rosa D; Cardelli, Luca; Csikász-Nagy, Attila
2018-06-18
Switch-like and oscillatory dynamical systems are widely observed in biology. We investigate the simplest biological switch that is composed of a single molecule that can be autocatalytically converted between two opposing activity forms. We test how this simple network can keep its switching behaviour under perturbations in the system. We show that this molecule can work as a robust bistable system, even for alterations in the reactions that drive the switching between various conformations. We propose that this single molecule system could work as a primitive biological sensor and show by steady state analysis of a mathematical model of the system that it could switch between possible states for changes in environmental signals. Particularly, we show that a single molecule phosphorylation-dephosphorylation switch could work as a nucleotide or energy sensor. We also notice that a given set of reductions in the reaction network can lead to the emergence of oscillatory behaviour. We propose that evolution could have converted this switch into a single molecule oscillator, which could have been used as a primitive timekeeper. We discuss how the structure of the simplest known circadian clock regulatory system, found in cyanobacteria, resembles the proposed single molecule oscillator. Besides, we speculate if such minimal systems could have existed in an RNA world.
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Llorach, Rafael; Garrido, Ignacio; Monagas, Maria; Urpi-Sarda, Mireia; Tulipani, Sara; Bartolome, Begona; Andres-Lacueva, Cristina
2010-11-05
Almond, as a part of the nut family, is an important source of biological compounds, and specifically, almond skins have been considered an important source of polyphenols, including flavan-3-ols and flavonols. Polyphenol metabolism may produce several classes of metabolites that could often be more biologically active than their dietary precursor and could also become a robust new biomarker of almond polyphenol intake. In order to study urinary metabolome modifications during the 24 h after a single dose of almond skin extract, 24 volunteers (n = 24), who followed a polyphenol-free diet for 48 h before and during the study, ingested a dietary supplement of almond skin phenolic compounds (n = 12) or a placebo (n = 12). Urine samples were collected before ((-2)-0 h) and after (0-2 h, 2-6 h, 6-10 h, and 10-24 h) the intake and were analyzed by liquid chromatography-mass spectrometry (LC-q-TOF) and multivariate statistical analysis (principal component analysis (PCA) and orthogonal projection to latent structures (OPLS)). Putative identification of relevant biomarkers revealed a total of 34 metabolites associated with the single dose of almond extract, including host and, in particular, microbiota metabolites. As far as we know, this is the first time that conjugates of hydroxyphenylvaleric, hydroxyphenylpropionic, and hydroxyphenylacetic acids have been identified in human samples after the consumption of flavan-3-ols through a metabolomic approach. The results showed that this non-targeted approach could provide new intake biomarkers, contributing to the development of the food metabolome as an important part of the human urinary metabolome.
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Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H Peter
2017-07-01
Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.
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Imaging and sizing of single DNA molecules on a mobile phone.
Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan Lok; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan
2014-12-23
DNA imaging techniques using optical microscopy have found numerous applications in biology, chemistry and physics and are based on relatively expensive, bulky and complicated set-ups that limit their use to advanced laboratory settings. Here we demonstrate imaging and length quantification of single molecule DNA strands using a compact, lightweight and cost-effective fluorescence microscope installed on a mobile phone. In addition to an optomechanical attachment that creates a high contrast dark-field imaging setup using an external lens, thin-film interference filters, a miniature dovetail stage and a laser-diode for oblique-angle excitation, we also created a computational framework and a mobile phone application connected to a server back-end for measurement of the lengths of individual DNA molecules that are labeled and stretched using disposable chips. Using this mobile phone platform, we imaged single DNA molecules of various lengths to demonstrate a sizing accuracy of <1 kilobase-pairs (kbp) for 10 kbp and longer DNA samples imaged over a field-of-view of ∼2 mm2.
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Trifunovic, Luka; Pedrocchi, Fabio L; Hoffman, Silas; Maletinsky, Patrick; Yacoby, Amir; Loss, Daniel
2015-06-01
Magnetic resonance techniques not only provide powerful imaging tools that have revolutionized medicine, but they have a wide spectrum of applications in other fields of science such as biology, chemistry, neuroscience and physics. However, current state-of-the-art magnetometers are unable to detect a single nuclear spin unless the tip-to-sample separation is made sufficiently small. Here, we demonstrate theoretically that by placing a ferromagnetic particle between a nitrogen-vacancy magnetometer and a target spin, the magnetometer sensitivity is improved dramatically. Using materials and techniques that are already experimentally available, our proposed set-up is sensitive enough to detect a single nuclear spin within ten milliseconds of data acquisition at room temperature. The sensitivity is practically unchanged when the ferromagnet surface to the target spin separation is smaller than the ferromagnet lateral dimensions; typically about a tenth of a micrometre. This scheme further benefits when used for nitrogen-vacancy ensemble measurements, enhancing sensitivity by an additional three orders of magnitude.
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Joint Identification of Genetic Variants for Physical Activity in Korean Population
Kim, Jayoun; Kim, Jaehee; Min, Haesook; Oh, Sohee; Kim, Yeonjung; Lee, Andy H.; Park, Taesung
2014-01-01
There has been limited research on genome-wide association with physical activity (PA). This study ascertained genetic associations between PA and 344,893 single nucleotide polymorphism (SNP) markers in 8842 Korean samples. PA data were obtained from a validated questionnaire that included information on PA intensity and duration. Metabolic equivalent of tasks were calculated to estimate the total daily PA level for each individual. In addition to single- and multiple-SNP association tests, a pathway enrichment analysis was performed to identify the biological significance of SNP markers. Although no significant SNP was found at genome-wide significance level via single-SNP association tests, 59 genetic variants mapped to 76 genes were identified via a multiple SNP approach using a bootstrap selection stability measure. Pathway analysis for these 59 variants showed that maturity onset diabetes of the young (MODY) was enriched. Joint identification of SNPs could enable the identification of multiple SNPs with good predictive power for PA and a pathway enriched for PA. PMID:25026172
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Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection.
De Paulis, Milena; Gilio, Alfredo Elias; Ferraro, Alexandre Archanjo; Ferronato, Angela Esposito; do Sacramento, Patrícia Rossi; Botosso, Viviane Fongaro; Oliveira, Danielle Bruna Leal de; Marinheiro, Juliana Cristina; Hársi, Charlotte Marianna; Durigon, Edison Luiz; Vieira, Sandra Elisabete
2011-01-01
To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.
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Study of Biological Pigments by Single Specimen Derivative Spectrophotometry
Goldstein, Jack M.
1970-01-01
The single specimen derivative (SSD) method provides an absolute absorption spectrum of a substance in the absence of a suitable reference. Both a reference and a measuring monochromatic beam pass through a single sample, and the specimen itself acts as its own reference. The two monochromatic beams maintain a fixed wavelength difference upon scanning, and the difference in absorbance of the two beams is determined. Thus, the resulting spectrum represents the first derivative of the conventional type absorption spectrum. Tissues and cell fractions have been examined at room and liquid N2 temperature and chromophoric molecules such as the mitochondrial cytochromes and blood pigments have been detectable in low concentrations. In the case of isolated cellular components, the observed effects of substrates and inhibitors confirm similar studies by conventional spectrophotometry. The extension of the SSD concept to the microscopic level has permitted the study of the tissue compartmentalization and function of cytochromes and other pigments within layered tissue. PMID:4392452
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NASA Astrophysics Data System (ADS)
Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H. Peter
2017-07-01
Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.
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Characterization of UVC-induced DNA damage in bloodstains: forensic implications.
Hall, Ashley; Ballantyne, Jack
2004-09-01
The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.
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Unsupervised multiple kernel learning for heterogeneous data integration.
Mariette, Jérôme; Villa-Vialaneix, Nathalie
2018-03-15
Recent high-throughput sequencing advances have expanded the breadth of available omics datasets and the integrated analysis of multiple datasets obtained on the same samples has allowed to gain important insights in a wide range of applications. However, the integration of various sources of information remains a challenge for systems biology since produced datasets are often of heterogeneous types, with the need of developing generic methods to take their different specificities into account. We propose a multiple kernel framework that allows to integrate multiple datasets of various types into a single exploratory analysis. Several solutions are provided to learn either a consensus meta-kernel or a meta-kernel that preserves the original topology of the datasets. We applied our framework to analyse two public multi-omics datasets. First, the multiple metagenomic datasets, collected during the TARA Oceans expedition, was explored to demonstrate that our method is able to retrieve previous findings in a single kernel PCA as well as to provide a new image of the sample structures when a larger number of datasets are included in the analysis. To perform this analysis, a generic procedure is also proposed to improve the interpretability of the kernel PCA in regards with the original data. Second, the multi-omics breast cancer datasets, provided by The Cancer Genome Atlas, is analysed using a kernel Self-Organizing Maps with both single and multi-omics strategies. The comparison of these two approaches demonstrates the benefit of our integration method to improve the representation of the studied biological system. Proposed methods are available in the R package mixKernel, released on CRAN. It is fully compatible with the mixOmics package and a tutorial describing the approach can be found on mixOmics web site http://mixomics.org/mixkernel/. jerome.mariette@inra.fr or nathalie.villa-vialaneix@inra.fr. Supplementary data are available at Bioinformatics online.
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A method for three-dimensional quantitative observation of the microstructure of biological samples
NASA Astrophysics Data System (ADS)
Wang, Pengfei; Chen, Dieyan; Ma, Wanyun; Wu, Hongxin; Ji, Liang; Sun, Jialin; Lv, Danyu; Zhang, Lu; Li, Ying; Tian, Ning; Zheng, Jinggao; Zhao, Fengying
2009-07-01
Contemporary biology has developed into the era of cell biology and molecular biology, and people try to study the mechanism of all kinds of biological phenomena at the microcosmic level now. Accurate description of the microstructure of biological samples is exigent need from many biomedical experiments. This paper introduces a method for 3-dimensional quantitative observation on the microstructure of vital biological samples based on two photon laser scanning microscopy (TPLSM). TPLSM is a novel kind of fluorescence microscopy, which has excellence in its low optical damage, high resolution, deep penetration depth and suitability for 3-dimensional (3D) imaging. Fluorescent stained samples were observed by TPLSM, and afterward the original shapes of them were obtained through 3D image reconstruction. The spatial distribution of all objects in samples as well as their volumes could be derived by image segmentation and mathematic calculation. Thus the 3-dimensionally and quantitatively depicted microstructure of the samples was finally derived. We applied this method to quantitative analysis of the spatial distribution of chromosomes in meiotic mouse oocytes at metaphase, and wonderful results came out last.
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Analysis of single mammalian cells on-chip.
Sims, Christopher E; Allbritton, Nancy L
2007-04-01
A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR.
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9 CFR 114.6 - Mixing biological products.
Code of Federal Regulations, 2013 CFR
2013-01-01
... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Mixing biological products. 114.6... BIOLOGICAL PRODUCTS § 114.6 Mixing biological products. Each biological product, when in liquid form, shall be mixed thoroughly in a single container. During bottling operations, the product shall be...
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9 CFR 114.6 - Mixing biological products.
Code of Federal Regulations, 2014 CFR
2014-01-01
... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Mixing biological products. 114.6... BIOLOGICAL PRODUCTS § 114.6 Mixing biological products. Each biological product, when in liquid form, shall be mixed thoroughly in a single container. During bottling operations, the product shall be...
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9 CFR 114.6 - Mixing biological products.
Code of Federal Regulations, 2011 CFR
2011-01-01
... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Mixing biological products. 114.6... BIOLOGICAL PRODUCTS § 114.6 Mixing biological products. Each biological product, when in liquid form, shall be mixed thoroughly in a single container. During bottling operations, the product shall be...
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9 CFR 114.6 - Mixing biological products.
Code of Federal Regulations, 2012 CFR
2012-01-01
... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Mixing biological products. 114.6... BIOLOGICAL PRODUCTS § 114.6 Mixing biological products. Each biological product, when in liquid form, shall be mixed thoroughly in a single container. During bottling operations, the product shall be...
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9 CFR 114.6 - Mixing biological products.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Mixing biological products. 114.6... BIOLOGICAL PRODUCTS § 114.6 Mixing biological products. Each biological product, when in liquid form, shall be mixed thoroughly in a single container. During bottling operations, the product shall be...
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Single ionization and capture cross sections from biological molecules by bare projectile impact*
NASA Astrophysics Data System (ADS)
Quinto, Michele A.; Monti, Juan M.; Montenegro, Pablo D.; Fojón, Omar A.; Champion, Christophe; Rivarola, Roberto D.
2017-02-01
We report calculations on single differential and total cross sections for single ionization and single electron capture from biological targets, namely, vapor water and DNA nucleobasese molecules, by bare projectile impact: H+, He2+, and C6+. They are performed within the Continuum Distorted Wave - Eikonal Initial State approximation and compared to several existing experimental data. This study is oriented to the obtention of a reliable set of theoretical data to be used as input in a Monte Carlo code destined to micro- and nano- dosimetry.
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Detection of small number of Giardia in biological materials prepared from stray dogs.
Esmailikia, Leila; Ebrahimzade, Elahe; Shayan, Parviz; Amininia, Narges
2017-12-20
Giardia lamblia is an intestinal protozoa with intermittent and low shedding especially in dogs, and the detection of Giardia is accompanied with problems such as sampling and diagnostic method. The objective of this study was to detection of Giardia in biological materials with low number of parasite using parasitological and molecular methods, and also to determine whether the examined stray dogs harbor known zoonotic genotype of Giardia. For this aim 85 fecal and duodenal samples were studied from which 1 was positive by Trichrome staining of stool, 4 were positive by staining of duodenal samples. The nested PCR analysis with primers derived from 18 SrRNA showed that the specific PCR product could be amplified in 4 stool and 4 duodenal samples. All positive samples in staining analysis were also positive in nested PCR. No amplification could be observed by nested PCR with primers derived from β giardin gene due to the single copy of gene. Interestingly, the extracted DNA from old fixed stained Giardia positive smears could be also amplified with primers derived from 18SrRNA gene. The sequence analysis of nested PCR products showed that they belong to the genotype D. In conclusion, it is to denote that the Trichrome or Giemsa methods were not suitable for the detection of small number of this parasite in stool and the nested PCR with primers derived from 18S rRNA gene can replace the traditional methods successfully. For detection of Giardia in stool, primers derived from β giardin will not be recommended.
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Multiple single-element transducer photoacoustic computed tomography system
NASA Astrophysics Data System (ADS)
Kalva, Sandeep Kumar; Hui, Zhe Zhi; Pramanik, Manojit
2018-02-01
Light absorption by the chromophores (hemoglobin, melanin, water etc.) present in any biological tissue results in local temperature rise. This rise in temperature results in generation of pressure waves due to the thermoelastic expansion of the tissue. In a circular scanning photoacoustic computed tomography (PACT) system, these pressure waves can be detected using a single-element ultrasound transducer (SUST) (while rotating in full 360° around the sample) or using a circular array transducer. SUST takes several minutes to acquire the PA data around the sample whereas the circular array transducer takes only a fraction of seconds. Hence, for real time imaging circular array transducers are preferred. However, these circular array transducers are custom made, expensive and not easily available in the market whereas SUSTs are cheap and readily available in the market. Using SUST for PACT systems is still cost effective. In order to reduce the scanning time to few seconds instead of using single SUST (rotating 360° ), multiple SUSTs can be used at the same time to acquire the PA data. This will reduce the scanning time by two-fold in case of two SUSTs (rotating 180° ) or by four-fold and eight-fold in case of four SUSTs (rotating 90° ) and eight SUSTs (rotating 45° ) respectively. Here we show that with multiple SUSTs, similar PA images (numerical and experimental phantom data) can be obtained as that of PA images obtained using single SUST.
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Modeling the genealogy of a cultural trait.
Aguilar, Elliot; Ghirlanda, Stefano
2015-05-01
The mathematical study of genealogies has yielded important insights in population biology, such as the ability to estimate the time to the most recent common ancestor (MRCA) of a sample of genetic sequences or of a group of individuals. Here we introduce a model of cultural genealogies that is a step toward answering similar questions for cultural traits. In our model individuals can inherit from a variable, potentially large number of ancestors, rather than from a fixed, small number of ancestors (one or two) as is typical of genetic evolution. We first show that, given a sample of individuals, a cultural common ancestor does not necessarily exist. We then introduce a related concept: the most recent unique ancestor (MRUA), i.e., the most recent single individual who is the earliest cultural ancestor of the sample. We show that, under neutral evolution, the time to the MRUA can be staggeringly larger than the time to MRCA in a single ancestor model, except when the average number of learning opportunities per individuals is small. Our results point out that the properties of cultural genealogies may be very different from those of genetic genealogies, with potential implications for reconstructing the histories of cultural traits. Copyright © 2014 Elsevier Inc. All rights reserved.
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Vidovic, Luka; Majaron, Boris
2014-02-01
Diffuse reflectance spectra (DRS) of biological samples are commonly measured using an integrating sphere (IS). To account for the incident light spectrum, measurement begins by placing a highly reflective white standard against the IS sample opening and collecting the reflected light. After replacing the white standard with the test sample of interest, DRS of the latter is determined as the ratio of the two values at each involved wavelength. However, such a substitution may alter the fluence rate inside the IS. This leads to distortion of measured DRS, which is known as single-beam substitution error (SBSE). Barring the use of more complex experimental setups, the literature states that only approximate corrections of the SBSE are possible, e.g., by using look-up tables generated with calibrated low-reflectivity standards. We present a practical method for elimination of SBSE when using IS equipped with an additional reference port. Two additional measurements performed at this port enable a rigorous elimination of SBSE. Our experimental characterization of SBSE is replicated by theoretical derivation. This offers an alternative possibility of computational removal of SBSE based on advance characterization of a specific DRS setup. The influence of SBSE on quantitative analysis of DRS is illustrated in one application example.
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Guan, Fuyu; Robinson, Mary A
2017-09-08
The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure. Both the theory and the developed SPE procedure have led to the development of a novel analytical method for comprehensive detection of multitudinous bioactive peptides in equine plasma and urine using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Thirty nine bioactive peptides were extracted with strong anion-exchange mixed-mode SPE sorbent, separated on a reversed-phase C 18 column and detected by HRMS and data-dependent tandem mass spectrometry. The limit of detection (LOD) was 10-50 pg mL -1 in plasma for most of the peptides and 100 pg mL -1 for the remaining. For urine, LOD was 20-400 pg mL -1 for most of the peptides and 1-4 ng mL -1 for the others. In vitro degradation of the peptides in equine plasma and urine was examined at ambient temperature; the peptides except those with a D-amino acid at position 2 were unstable not only in plasma but also in urine. The developed method was successful in analysis of plasma and urine samples from horses administered dermorphin. Additionally, dermorphin metabolites were identified in the absence of reference standards. The developed SPE procedure and LC-HRMS method can theoretically detect virtually all peptides present at a sufficient concentration in a sample. New peptides can be readily included in the method to be detected without method re-development. The developed method also generates such data that can be retrospectively analyzed for peptides unknown at the time of sample analysis. It is the first generic analytical method for comprehensive detection of multitudinous exogenous peptides in biological samples, to the authors' knowledge. Copyright © 2017 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Girkin, John M.; Burns, David; Dawson, Martin D.
1999-06-01
We report on the development of practical and user friendly lasers for multiphoton imaging of biological material. The laser developed for the work is a laser diode pumped Cr:LiSAF source modelocked using a saturable Bragg reflector as the passive modelocking element. For this system we routinely obtain 100 fs pulses at a repetition rate 200 MHz with an average output power of 20 mW. The laser has a single operator control and is particularly suitable for use by non-laser specialists. We have used the source developed to image a range of biologically significant samples. The initial work has centered on the imaging of intact human dental tissue. The first two-photon images of dental tissue are reported showing the development of early dental disease from depths up to 500 micrometers into the tooth. These results demonstrate the detection of carious lesions before the more conventional techniques currently used by dental practitioners. Work on other living intact biological tissue is also reported, in particular plants containing a genetically bred fluorescent marker to enable the examination of complete and intact living plant tissue.
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Dick, Jeffrey E.; Hilterbrand, Adam T.; Boika, Aliaksei; Upton, Jason W.; Bard, Allen J.
2015-01-01
We report observations of stochastic collisions of murine cytomegalovirus (MCMV) on ultramicroelectrodes (UMEs), extending the observation of discrete collision events on UMEs to biologically relevant analytes. Adsorption of an antibody specific for a virion surface glycoprotein allowed differentiation of MCMV from MCMV bound by antibody from the collision frequency decrease and current magnitudes in the electrochemical collision experiments, which shows the efficacy of the method to size viral samples. To add selectivity to the technique, interactions between MCMV, a glycoprotein-specific primary antibody to MCMV, and polystyrene bead “anchors,” which were functionalized with a secondary antibody specific to the Fc region of the primary antibody, were used to affect virus mobility. Bead aggregation was observed, and the extent of aggregation was measured using the electrochemical collision technique. Scanning electron microscopy and optical microscopy further supported aggregate shape and extent of aggregation with and without MCMV. This work extends the field of collisions to biologically relevant antigens and provides a novel foundation upon which qualitative sensor technology might be built for selective detection of viruses and other biologically relevant analytes. PMID:25870261
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Multistrip western blotting to increase quantitative data output.
Kiyatkin, Anatoly; Aksamitiene, Edita
2009-01-01
The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.