Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens

PubMed Central

Glaser, Adam K.; Reder, Nicholas P.; Chen, Ye; McCarty, Erin F.; Yin, Chengbo; Wei, Linpeng; Wang, Yu; True, Lawrence D.; Liu, Jonathan T.C.

2017-01-01

For the 1.7 million patients per year in the U.S. who receive a new cancer diagnosis, treatment decisions are largely made after a histopathology exam. Unfortunately, the gold standard of slide-based microscopic pathology suffers from high inter-observer variability and limited prognostic value due to sampling limitations and the inability to visualize tissue structures and molecular targets in their native 3D context. Here, we show that an open-top light-sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. We demonstrate the utility of this technology for various applications: wide-area surface microscopy to triage surgical specimens (with ~200 μm surface irregularities), rapid intraoperative assessment of tumour-margin surfaces (12.5 sec/cm2), and volumetric assessment of optically cleared core–needle biopsies (1 mm in diameter, 2 cm in length). Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens. PMID:29750130

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

PubMed Central

Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

2016-01-01

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

Rapid identification of Salmonella serotypes through hyperspectral microscopy with different lighting sources

USDA-ARS?s Scientific Manuscript database

Hyperspectral microscope imaging (HMI) has the potential to classify foodborne pathogenic bacteria at cell level by combining microscope images with a spectrophotometer. In this study, the spectra generated from HMIs of five live Salmonella serovars from two light sources, metal halide (MH) and tun...

Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

USGS Publications Warehouse

Walker, G.K.; Black, M.G.; Edwards, C.A.

1996-01-01

Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

Structured light optical microscopy for three-dimensional reconstruction of technical surfaces

NASA Astrophysics Data System (ADS)

Kettel, Johannes; Reinecke, Holger; Müller, Claas

2016-04-01

In microsystems technology quality control of micro structured surfaces with different surface properties is playing an ever more important role. The process of quality control incorporates three-dimensional (3D) reconstruction of specularand diffusive reflecting technical surfaces. Due to the demand on high measurement accuracy and data acquisition rates, structured light optical microscopy has become a valuable solution to solve this problem providing high vertical and lateral resolution. However, 3D reconstruction of specular reflecting technical surfaces still remains a challenge to optical measurement principles. In this paper we present a measurement principle based on structured light optical microscopy which enables 3D reconstruction of specular- and diffusive reflecting technical surfaces. It is realized using two light paths of a stereo microscope equipped with different magnification levels. The right optical path of the stereo microscope is used to project structured light onto the object surface. The left optical path is used to capture the structured illuminated object surface with a camera. Structured light patterns are generated by a Digital Light Processing (DLP) device in combination with a high power Light Emitting Diode (LED). Structured light patterns are realized as a matrix of discrete light spots to illuminate defined areas on the object surface. The introduced measurement principle is based on multiple and parallel processed point measurements. Analysis of the measured Point Spread Function (PSF) by pattern recognition and model fitting algorithms enables the precise calculation of 3D coordinates. Using exemplary technical surfaces we demonstrate the successful application of our measurement principle.

Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.

PubMed

Amat, Fernando; Keller, Philipp J

2013-05-01

Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions. Copyright © 2013 S. Karger AG, Basel.

Intraoperative Fluorescence Cerebral Angiography by Laser Surgical Microscopy: Comparison With Xenon Microscopy and Simultaneous Observation of Cerebral Blood Flow and Surrounding Structures.

PubMed

Ito, Yuhei; Suzuki, Kyouichi; Ichikawa, Tsuyoshi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2018-06-12

Laser surgical microscopes should enable uniform illumination of the operative field, and require less luminous energy compared with existing xenon surgical microscopes. To examine the utility of laser illumination in fluorescence cerebral angiography. Fluorescein sodium (fluorescein) was used as a fluorescent dye. We first compared the clarity of cerebral blood flow images collected by fluorescence angiography between the laser illumination and xenon illumination methods. We then assessed use of the laser illuminator for simultaneous observation of blood flow and surrounding structures during fluorescence angiography. Furthermore, the study was designed to evaluate usefulness of the thus determined excitation light in clinical cases. Fluorescence angiography using blue light laser for excitation provided higher clarity and contrast blood flow images compared with using blue light generated from a xenon lamp. Further, illumination with excitation light consisting of a combination of 3 types of laser (higher level of blue light, no green light, and lower level of red light) enabled both blood flow and surrounding structures to be observed through the microscope directly by the surgeon. Laser-illuminated fluorescence angiography provides high clarity and contrast images of cerebral blood flow. Further, a laser providing strong blue light and weak red light for excitation light enables simultaneous visual observation of fluorescent blood flow and surrounding structures by the surgeon using a surgical microscope. Overall, these data suggest that laser surgical microscopes are useful for both ordinary operative manipulations and fluorescence angiography.

Innovative Strategies for Clinical Microscopy Instruction: Virtual Versus Light Microscopy.

PubMed

McDaniel, M Jane; Russell, Gregory B; Crandall, Sonia J

2018-06-01

The purpose of the study was to compare virtual microscopy with light microscopy to determine differences in learning outcomes and learner attitudes in teaching clinical microscopy to physician assistant (PA) students. A prospective, randomized, crossover design study was conducted with a convenience sample of 67 first-year PA students randomized to 2 groups. One group used light microscopes to find microscopic structures, whereas the other group used instructor-directed video streaming of microscopic elements. At the midpoint of the study, the groups switched instructional strategies. Learning outcomes were assessed via posttest after each section of the study, with comparison of final practical examination results to previous cohorts. Attitudes about the 2 educational strategies were assessed through a postcourse questionnaire with a Likert scale. Analysis of the first posttest demonstrated that students in the video-streamed group had significantly better learning outcomes than those in the light microscopy group (P = .004; Cohen's d = 0.74). Analysis of the posttest after crossover showed no differences between the 2 groups (P = .48). Between the 2 posttests, students first assigned to the light microscopy group scored a 6.6 mean point increase (±10.4 SD; p = .0011), whereas students first assigned to the virtual microscopy group scored a 1.3 mean point increase (±7.1 SD; p = .29). The light microscopy group improved more than the virtual microscopy group (P = .019). Analysis of practical examination data revealed higher scores for the study group compared with 5 previous cohorts of first-year students (P < .0001; Cohen's d = 0.66). Students preferred virtual microscopy to traditional light microscopy. Virtual microscopy is an effective educational strategy, and students prefer this method when learning to interpret images of clinical specimens.

Evaluation of laser ablation microtomy for correlative microscopy of hard tissues.

PubMed

Boyde, A

2018-02-27

Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly-methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE-SEM and confocal scanning light microscopy (CSLM). Most were also studied by X-yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near-infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1-2 μm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the 'voxel' in most laboratory X-ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Restoration of uneven illumination in light sheet microscopy images.

PubMed

Uddin, Mohammad Shorif; Lee, Hwee Kuan; Preibisch, Stephan; Tomancak, Pavel

2011-08-01

Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images.

Concepts in Light Microscopy of Viruses

PubMed Central

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Concepts in Light Microscopy of Viruses.

PubMed

Witte, Robert; Andriasyan, Vardan; Georgi, Fanny; Yakimovich, Artur; Greber, Urs F

2018-04-18

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

AccessScope project: Accessible light microscope for users with upper limb mobility or visual impairments.

PubMed

Mansoor, Awais; Ahmed, Wamiq M; Samarapungavan, Ala; Cirillo, John; Schwarte, David; Robinson, J Paul; Duerstock, Bradley S

2010-01-01

A web-based application was developed to remotely view slide specimens and control all functions of a research-level light microscopy workstation, called AccessScope. Students and scientists with upper limb mobility and visual impairments are often unable to use a light microscope by themselves and must depend on others in its operation. Users with upper limb mobility impairments and low vision were recruited to assist in the design process of the AccessScope personal computer (PC) user interface. Participants with these disabilities were evaluated in their ability to use AccessScope to perform microscopical tasks. AccessScope usage was compared with inspecting prescanned slide images by grading participants' identification and understanding of histological features and knowledge of microscope operation. With AccessScope subjects were able to independently perform common light microscopy functions through an Internet browser by employing different PC pointing devices or accessibility software according to individual abilities. Subjects answered more histology and microscope usage questions correctly after first participating in an AccessScope test session. AccessScope allowed users with upper limb or visual impairments to successfully perform light microscopy without assistance. This unprecedented capability is crucial for students and scientists with disabilities to perform laboratory coursework or microscope-based research and pursue science, technology, engineering, and mathematics fields.

Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

PubMed Central

Sivaguru, Mayandi; Mander, Luke; Fried, Glenn; Punyasena, Surangi W.

2012-01-01

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed. PMID:22720050

Light-induced c-Fos expression in the mouse suprachiasmatic nucleus: immunoelectron microscopy reveals co-localization in multiple cell types.

PubMed

Castel, M; Belenky, M; Cohen, S; Wagner, S; Schwartz, W J

1997-09-01

Although light is known to regulate the level of c-fos gene expression in the suprachiasmatic nucleus (SCN), the site of an endogenous circadian clock, little is known about the identities of the photically activated cells. We used light-microscopic immunocytochemistry and immunoelectron microscopy to detect c-Fos protein in the SCN of Sabra mice exposed to brief nocturnal light pulses at zeitgeber time 15-16. Stimulation with light pulses that saturated the phase-shifting response of the circadian locomotor rhythm revealed an upper limit to the number of photo-inducible c-Fos cells at about one-fifth of the estimated total SCN cell population. This functionally defined set was morphologically and phenotypically heterogeneous. About 24% could be labelled for vasoactive intestinal polypeptide, 13% for vasopressin-neurophysin, and 7% for glial fibrillary acidic protein. The remaining 56% of c-Fos-positive cells were largely of unknown phenotype, although many were presumptive interneurons, some of which were immunoreactive for nitric oxide synthase.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

PubMed Central

Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

Contributed review: Review of integrated correlative light and electron microscopy.

PubMed

Timmermans, F J; Otto, C

2015-01-01

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

PubMed

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Tissue and cellular localization of tannins in Tunisian dates (Phoenix dactylifera L.) by light and transmission electron microscopy.

PubMed

Hammouda, Hédi; Alvarado, Camille; Bouchet, Brigitte; Kalthoum-Chérif, Jamila; Trabelsi-Ayadi, Malika; Guyot, Sylvain

2014-07-16

A histological approach including light microscopy and transmission electron microscopy (TEM) was used to provide accurate information on the localization of condensed tannins in the edible tissues and in the stone of date fruits (Phoenix dactylifera L.). Light microscopy was carried out on fresh tissues after staining by 4-dimethylaminocinnamaldehyde (DMACA) for a specific detection of condensed tannins. Thus, whether under light microscopy or transmission electron microscopy (TEM), results showed that tannins are not located in the epidermis but more deeply in the mesocarp in the vacuole of very large cells. Regarding the stones, tannins are found in a specific cell layer located at 50 μm from the sclereid cells of the testa.

Reduction of parasitic interferences in digital holographic microscopy by numerically decreased coherence length

NASA Astrophysics Data System (ADS)

Kosmeier, S.; Langehanenberg, P.; von Bally, G.; Kemper, B.

2012-01-01

Due to the large coherence length of laser light, optical path length (OPL) resolution in laser based digital holographic microscopy suffers from parasitic interferences caused by multiple reflections within the experimental setup. Use of partially coherent light reduces this drawback but requires precise and stable matching of object and reference arm's OPLs and limits the spatial frequency of the interference pattern in off-axis holography. Here, we investigate if the noise properties of spectrally broadened light sources can be generated numerically. Therefore, holograms are coherently captured at different laser wavelengths and the corresponding reconstructed wave fields are numerically superimposed utilizing variable weightings. Gaussian and rectangular spectral shapes of the so synthesized field are analyzed with respect to the resulting noise level, which is quantified in OPL distributions of a reflective test target. Utilizing a Gaussian weighting, the noise level is found to be similar to the one obtained with the partially coherent light of a superluminescent diode. With a rectangular shaped synthesized spectrum, noise is reduced more efficient than with a Gaussian one. The applicability of the method in label-free cell analysis is demonstrated by quantitative phase contrast images obtained from living cancer cells.

Hybrid of two-photon microscopy and optical multimodality imaging for multi-scale imaging of small animals

NASA Astrophysics Data System (ADS)

Li, Tianmeng; Hui, Hui; Ma, He; Yang, Xin; Tian, Jie

2018-02-01

Non-invasive imaging technologies, such as magnetic resonance imaging (MRI) and optical multimodality imaging methods, are commonly used for diagnosing and supervising the development of inflammatory bowel disease (IBD). These in vivo imaging methods can provide morphology changes information of IBD in macro-scale. However, it is difficult to investigate the intestinal wall in molecular and cellular level. State-of-art light-sheet and two-photon microscopy have the ability to acquire the changes for IBD in micro-scale. The aim of this work is to evaluate the size of the enterocoel and the thickness of colon wall using both MRI for in vivo imaging, and light-sheet and two-photon microscope for in vitro imaging. C57BL/6 mice were received 3.5% Dextran sodium sulfate (DSS) in the drinking water for 5 days to build IBD model. Mice were imaged with MRI on days 0, 6 to observe colitis progression. After MRI imaging, the mice were sacrificed to take colons for tissue clearing. Then, light-sheet and two-photon microscopies are used for in vitro imaging of the cleared samples. The experimental group showed symptoms of bloody stools, sluggishness and weight loss. It showed that the colon wall was thicker while the enterocoel was narrower compare to control group. The more details are observed using light-sheet and two-photon microscope. It is demonstrated that hybrid of MRI in macro-scale and light-sheet and two-photon microscopy in micro-scale imaging is feasible for colon inflammation diagnosing and supervising.

Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

NASA Astrophysics Data System (ADS)

Hashim, Fatimah; Amin, Nakisah Mat

2017-02-01

Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

A new microscope optics for laser dark-field illumination applied to high precision two dimensional measurement of specimen displacement.

PubMed

Noda, Naoki; Kamimura, Shinji

2008-02-01

With conventional light microscopy, precision in the measurement of the displacement of a specimen depends on the signal-to-noise ratio when we measure the light intensity of magnified images. This implies that, for the improvement of precision, getting brighter images and reducing background light noise are both inevitably required. For this purpose, we developed a new optics for laser dark-field illumination. For the microscopy, we used a laser beam and a pair of axicons (conical lenses) to get an optimal condition for dark-field observations. The optics was applied to measuring two dimensional microbead displacements with subnanometer precision. The bandwidth of our detection system overall was 10 kHz. Over most of this bandwidth, the observed noise level was as small as 0.1 nm/radicalHz.

Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

NASA Astrophysics Data System (ADS)

Christensen, A. Kent; Lowry, Terry B.

1995-10-01

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about [minus sign]117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at [minus sign]155 to [minus sign]170°C to produce semithin frozen sections (0.5 to 3 [mu]m thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

A simple procedure to analyze positions of interest in infectious cell cultures by correlative light and electron microscopy.

PubMed

Madela, Kazimierz; Banhart, Sebastian; Zimmermann, Anja; Piesker, Janett; Bannert, Norbert; Laue, Michael

2014-01-01

Plastic cell culture dishes that contain a thin bottom of highest optical quality including an imprinted finder grid (μ-Dish Grid-500) are optimally suited for routine correlative light and electron microscopy using chemical fixation. Such dishes allow high-resolution fluorescence and bright-field imaging using fixed and living cells and are compatible with standard protocols for scanning and transmission electron microscopy. Ease of use during cell culture and imaging, as well as a tight cover render the dishes particularly suitable for working with infectious organisms up to the highest biosafety level. Detailed protocols are provided and demonstrated by showing two examples: monitoring the production of virus-like particles of the Human Endogenous Retrovirus HERV-K(HML-2) by HeLa cells and investigation of Rab11-positive membrane-compartments of HeLa cells after infection with Chlamydia trachomatis. © 2014 Elsevier Inc. All rights reserved.

Investigating portable fluorescent microscopy (CyScope) as an alternative rapid diagnostic test for malaria in children and women of child-bearing age.

PubMed

Sousa-Figueiredo, José Carlos; Oguttu, David; Adriko, Moses; Besigye, Fred; Nankasi, Andrina; Arinaitwe, Moses; Namukuta, Annet; Betson, Martha; Kabatereine, Narcis B; Stothard, J Russell

2010-08-27

Prompt and correct diagnosis of malaria is crucial for accurate epidemiological assessment and better case management, and while the gold standard of light microscopy is often available, it requires both expertise and time. Portable fluorescent microscopy using the CyScope offers a potentially quicker, easier and more field-applicable alternative. This article reports on the strengths, limitations of this methodology and its diagnostic performance in cross-sectional surveys on young children and women of child-bearing age. 552 adults (99% women of child-bearing age) and 980 children (99% ≤ 5 years of age) from rural and peri-urban regions of Ugandan were examined for malaria using light microscopy (Giemsa-stain), a lateral-flow test (Paracheck-Pf) and the CyScope. Results from the surveys were used to calculate diagnostic performance (sensitivity and specificity) as well as to perform a receiver operating characteristics (ROC) analyses, using light microscopy as the gold-standard. Fluorescent microscopy (qualitative reads) showed reduced specificity (<40%), resulting in higher community prevalence levels than those reported by light microscopy, particularly in adults (+180% in adults and +20% in children). Diagnostic sensitivity was 92.1% in adults and 86.7% in children, with an area under the ROC curve of 0.63. Importantly, optimum performance was achieved for higher parasitaemia (>400 parasites/μL blood): sensitivity of 64.2% and specificity of 86.0%. Overall, the diagnostic performance of the CyScope was found inferior to that of Paracheck-Pf. Fluorescent microscopy using the CyScope is certainly a field-applicable and relatively affordable solution for malaria diagnoses especially in areas where electrical supplies may be lacking. While it is unlikely to miss higher parasitaemia, its application in cross-sectional community-based studies leads to many false positives (i.e. small fluorescent bodies of presently unknown origin mistaken as malaria parasites). Without recourse to other technologies, arbitration of these false positives is presently equivocal, which could ultimately lead to over-treatment; something that should be further explored in future investigations if the CyScope is to be more widely implemented.

Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: III. Correlative Light and Electron Microscopic Study

PubMed Central

Onouchi, Takanori; Shiogama, Kazuya; Mizutani, Yasuyoshi; Takaki, Takashi; Tsutsumi, Yutaka

2016-01-01

Neutrophil extracellular traps (NETs) released from dead neutrophils at the site of inflammation represent webs of neutrophilic DNA stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. We have shown the coexistence of NETs and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. In the present study, correlative light and electron microscopy (CLEM) employing confocal laser scanning microscopy and scanning electron microscopy was performed to bridge light and electron microscopic images of NETs and fibrin fibrils in formalin-fixed, paraffin-embedded, autopsied lung sections of legionnaire’s pneumonia. Lactoferrin immunoreactivity and 4'-6-diamidino-2-phenylindole (DAPI) reactivity were used as markers of NETs, and fibrin was probed by fibrinogen gamma chain. Of note is that NETs light microscopically represented as lactoferrin and DAPI-colocalized dots, 2.5 μm in diameter. CLEM gave super-resolution images of NETs and fibrin fibrils: “Dotted” NETs were ultrastructurally composed of fine filaments and masses of 58 nm-sized globular materials. A fibrin fibril consisted of clusters of smooth-surfaced filaments. NETs filaments (26 nm in diameter) were significantly thinner than fibrin filaments (295 nm in diameter). Of note is that CLEM was applicable to formalin-fixed, paraffin-embedded sections of autopsy material. PMID:27917008

Williams configures the LMM

NASA Image and Video Library

2016-04-18

ISS047e066551 (04/18/2016) --- NASA astronaut Jeff Williams configures the station’s Light Microscopy Module (LMM), a modified commercial, highly flexible, state-of-the-art light imaging microscope facility that provides researchers with powerful diagnostic hardware and software. The LMM enables novel research of microscopic phenomena in microgravity, with the capability of remotely acquiring and downloading digital images and videos across many levels of magnification.

The e-evolution of microscopy in dental education.

PubMed

Farah, Camile S; Maybury, Terrence S

2009-08-01

Recent technological innovation has now made it possible to turn the computer into a microscope. This has entailed a shift from light microscopy to virtual microscopy. This development then foregrounds the issue of the pedagogy involved in this move from the analogue technology of the light microscope to the digital, computerized instance of virtual microscopy. In order to address this issue, undergraduate students enrolled in the Bachelor of Dental Science program at the University of Queensland School of Dentistry were surveyed to ascertain their preference for light or virtual microscopy. The value of this study is that it was conducted on the same cohort of students in two separate courses in 2006 and 2008, giving it longitudinal validity. The responses were overwhelmingly in favor of virtual microscopy. When it came to completely replacing the light microscope with virtual microscopy, however, students were much more ambivalent about such a wholesale change although this was less of an issue in the senior year. This shift from light to virtual microscopy signals larger changes in the tertiary sector from print-literate to electronic forms of knowledge and from teacher-centered to student-focused frames of learning. In short, we are in the midst of the e-evolution of microscopy in dental education.

The role of light microscopy in aerospace analytical laboratories

NASA Technical Reports Server (NTRS)

Crutcher, E. R.

1977-01-01

Light microscopy has greatly reduced analytical flow time and added new dimensions to laboratory capability. Aerospace analytical laboratories are often confronted with problems involving contamination, wear, or material inhomogeneity. The detection of potential problems and the solution of those that develop necessitate the most sensitive and selective applications of sophisticated analytical techniques and instrumentation. This inevitably involves light microscopy. The microscope can characterize and often identify the cause of a problem in 5-15 minutes with confirmatory tests generally less than one hour. Light microscopy has and will make a very significant contribution to the analytical capabilities of aerospace laboratories.

[Clinical pathology on the verge of virtual microscopy].

PubMed

Tolonen, Teemu; Näpänkangas, Juha; Isola, Jorma

2015-01-01

For more than 100 years, examinations of pathology specimens have relied on the use of the light microscope. The technological progress of the last few years is enabling the digitizing of histologic specimen slides and application of the virtual microscope in diagnostics. Virtual microscopy will facilitate consultation possibilities, and digital image analysis serves to enhance the level of diagnostics. Organizing and monitoring clinicopathological meetings will become easier. Digital archive of histologic specimens and the virtual microscopy network are expected to benefit training and research as well, particularly what applies to the Finnish biobank network which is currently being established.

Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Mammalian Brain.

PubMed

Cristino, Luigia; Imperatore, Roberta; Di Marzo, Vincenzo

2017-01-01

This chapter attempts to piece together knowledge about new advanced microscopy techniques to study the neuroanatomical distribution of endocannabinoid receptors and enzymes at the level of cellular and subcellular structures and organelles in the brain. Techniques ranging from light to electron microscopy up to the new advanced LBM, PALM, and STORM super-resolution microscopy will be discussed in the context of their contribution to define the spatial distribution and organization of receptors and enzymes of the endocannabinoid system (ECS), and to better understand ECS brain functions. © 2017 Elsevier Inc. All rights reserved.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells.

PubMed

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2013-01-01

Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

PubMed Central

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2016-01-01

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

Single-molecule fluorescence microscopy review: shedding new light on old problems

PubMed Central

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

Light sheet microscopy.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail. © 2014 Elsevier Inc. All rights reserved.

Correlation of two-photon in vivo imaging and FIB/SEM microscopy

PubMed Central

Blazquez-Llorca, L; Hummel, E; Zimmerman, H; Zou, C; Burgold, S; Rietdorf, J; Herms, J

2015-01-01

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two-photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long-term two-photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer’s disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three-dimensional high-resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system. Lay Description Neuroscience and the understanding of brain functions are closely linked to the technical advances in microscopy. In this study we performed a correlative microscopy technique that offers the possibility to combine 2 photon in vivo imaging and FIB/SEM microscopy. Long term 2 photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool to study the dynamics of neurodegenerative diseases, such as Alzheimer’s disease. However, light microscopy has important limitations in revealing synapses that are the connections between neurons, and for this purpose, the electron microscopy is necessary. FIB/SEM microscopy is a novel tool for three-dimensional (3D) high resolution reconstructions since it acquires automated serial images at ultrastructural level. This correlative technique will open up new horizons and opportunities for unravelling the complexity of the nervous system. PMID:25786682

Introduction to Modern Methods in Light Microscopy.

PubMed

Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

2017-01-01

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

Effects of microgravity on muscle and cerebral cortex: a suggested interaction

NASA Astrophysics Data System (ADS)

D'Amelio, F.; Fox, R. A.; Wu, L. C.; Daunton, N. G.; Corcoran, M. L.

The ``slow'' antigravity muscle adductor longus was studied in rats after 14 days of spaceflight (SF). The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light and electron microscopy revealed myofiber atrophy, segmental necrosis and regenerative myofibers. Regenerative myofibers were N-CAM immunoreactive (N-CAM-IR). The neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles, degenerative changes, vacant axonal spaces and changes suggestive of axonal sprouting. No alterations of muscle spindles was seen either by light or electron microscopy. These observations suggest that muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight. In a separate study, GABA immunoreactivity (GABA-IR) was evaluated at the level of the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension (``simulated'' microgravity). A reduction in number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-IR terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system after spaceflight and hindlimb suspension it is suggested that after limb unloading there are alterations of afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the changes observed in GABA immunoreactivity of cells and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.

Integration of a high-NA light microscope in a scanning electron microscope.

PubMed

Zonnevylle, A C; Van Tol, R F C; Liv, N; Narvaez, A C; Effting, A P J; Kruit, P; Hoogenboom, J P

2013-10-01

We present an integrated light-electron microscope in which an inverted high-NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub-10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum-compatible immersion oil. For a 40-nm-diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Biobeam—Multiplexed wave-optical simulations of light-sheet microscopy

PubMed Central

Weigert, Martin; Bundschuh, Sebastian T.

2018-01-01

Sample-induced image-degradation remains an intricate wave-optical problem in light-sheet microscopy. Here we present biobeam, an open-source software package that enables simulation of operational light-sheet microscopes by combining data from 105–106 multiplexed and GPU-accelerated point-spread-function calculations. The wave-optical nature of these simulations leads to the faithful reproduction of spatially varying aberrations, diffraction artifacts, geometric image distortions, adaptive optics, and emergent wave-optical phenomena, and renders image-formation in light-sheet microscopy computationally tractable. PMID:29652879

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Light Microscopy Module Fan Disturbance Characterized Through Microgravity Emissions Laboratory Testing

NASA Technical Reports Server (NTRS)

McNelis, Anne M.; Motil, Susan M.

2003-01-01

A Light Microscopy Module (LMM) is being engineered, designed, and developed at the NASA Glenn Research Center. The LMM is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and control of physical science and biological science experiments within Glenn s Fluids Integrated Rack on the International Space Station. The LMM concept is a modified commercial research imaging light microscope with powerful laser-diagnostic hardware and interfaces, creating a one-of-a-kind, state-of-the-art microscopic research facility. The microscope will house several different objectives, corresponding to magnifications of 10, 40, 50, 63, and 100. Features of the LMM include high-resolution color video microscopy, brightfield, darkfield, phase contrast, differential interference contrast, spectrophotometry, and confocal microscopy combined in a single configuration. Also, laser tweezers are integrated with the diagnostics as a sample manipulation technique. As part of the development phase of the LMM, it was necessary to quantify the microgravity disturbances generated by the control box fan. Isolating the fan was deemed necessary to reduce the fan speed harmonic amplitudes and to eliminate any broadband disturbances across the 60- to 70-Hz and 160- to 170-Hz frequency ranges. The accelerations generated by a control box fan component of the LMM were measured in the Microgravity Emissions Laboratory (MEL). The MEL is a low-frequency measurement system developed to simulate and verify the on-orbit International Space Station (ISS) microgravity environment. The accelerations generated by various operating components of the ISS, if too large, could hinder the science performed onboard by disturbing the microgravity environment. The MEL facility gives customers a test-verified way of measuring their compliance with ISS limitations on vibratory disturbance levels. The facility is unique in that inertial forces in 6 degrees of freedom can be characterized simultaneously for an operating test article. Vibratory disturbance levels are measured for engineering or flight-level hardware following development from component to subassembly through the rack-level configuration. The MEL can measure accelerations as small as 10-7g, the accuracy needed to confirm compliance with ISS requirements.

A comparison of honey bee-collected pollen from working agricultural lands using light microscopy and ITS metabarcoding

USGS Publications Warehouse

Smart, Matthew; Cornman, Robert S.; Iwanowicz, Deborah; McDermott-Kubeczko, Margaret; Pettis, Jeff S; Spivak, Marla S; Otto, Clint R.

2017-01-01

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010–2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts.

Dual-dimensional microscopy: real-time in vivo three-dimensional observation method using high-resolution light-field microscopy and light-field display.

PubMed

Kim, Jonghyun; Moon, Seokil; Jeong, Youngmo; Jang, Changwon; Kim, Youngmin; Lee, Byoungho

2018-06-01

Here, we present dual-dimensional microscopy that captures both two-dimensional (2-D) and light-field images of an in-vivo sample simultaneously, synthesizes an upsampled light-field image in real time, and visualizes it with a computational light-field display system in real time. Compared with conventional light-field microscopy, the additional 2-D image greatly enhances the lateral resolution at the native object plane up to the diffraction limit and compensates for the image degradation at the native object plane. The whole process from capturing to displaying is done in real time with the parallel computation algorithm, which enables the observation of the sample's three-dimensional (3-D) movement and direct interaction with the in-vivo sample. We demonstrate a real-time 3-D interactive experiment with Caenorhabditis elegans. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Virtual Microscopy in Histopathology Training: Changing Student Attitudes in 3 Successive Academic Years.

PubMed

Bertram, Christof A; Firsching, Theresa; Klopfleisch, Robert

2018-01-01

Several veterinary faculties have integrated virtual microscopy into their curricula in recent years to improve and refine their teaching techniques. The many advantages of this recent technology are described in the literature, including remote access and an equal and constant slide quality for all students. However, no study has analyzed the change of perception toward virtual microscopy at different time points of students' academic educations. In the present study, veterinary students in 3 academic years were asked for their perspectives and attitudes toward virtual microscopy and conventional light microscopy. Third-, fourth-, and fifth-year veterinary students filled out a questionnaire with 12 questions. The answers revealed that virtual microscopy was overall well accepted by students of all academic years. Most students even suggested that virtual microscopy be implemented more extensively as the modality for final histopathology examinations. Nevertheless, training in the use of light microscopy and associated skills was surprisingly well appreciated. Regardless of their academic year, most students considered these skills important and necessary, and they felt that light microscopy should not be completely replaced. The reasons for this view differed depending on academic year, as the perceived main disadvantage of virtual microscopy varied. Third-year students feared that they would not acquire sufficient light microscopy skills. Fifth-year students considered technical difficulties (i.e., insufficient transmission speed) to be the main disadvantage of this newer teaching modality.

Sample holder for axial rotation of specimens in 3D microscopy.

PubMed

Bruns, T; Schickinger, S; Schneckenburger, H

2015-10-01

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

You can't measure what you can't see - detectors for microscopies

NASA Astrophysics Data System (ADS)

Denes, Peter

For centuries, the human eye has been the imaging detector of choice thanks to its high sensitivity, wide dynamic range, and direct connection to a built-in data recording and analysis system. The eye, however, is limited to visible light, which excludes microscopies with electrons and X-rays, and the built-in recording system stores archival information at very low rates. The former limitation has been overcome by ``indirect'' detectors, which convert probe particles to visible light, and the latter by a variety of recording techniques, from photographic film to semiconductor-based imagers. Semiconductor imagers have been used for decades as ``direct'' detectors in particle physics, and almost as long for hard X-rays. For soft X-ray microscopy, the challenge has been the small signal levels - plus getting the X-rays into the detector itself, given how quickly they are absorbed in inert layers. For electron microscopy, the challenge has been reconciling detector spatial resolution and pixel count with the large multiple scattering of electrons with energies used for microscopy. Further, a high recording rate (``movies'' rather than ``snapshots'') enables time-resolved studies, time-dependent corrections, shot-by-shot experiments and scanning techniques - at the expense of creating large data volumes. This talk will discuss solutions to these challenges, as well as an outlook towards future developments.

Impact of gluten-friendly™ technology on wheat kernel endosperm and gluten protein structure in seeds by light and electron microscopy.

PubMed

Landriscina, L; D'Agnello, P; Bevilacqua, A; Corbo, M R; Sinigaglia, M; Lamacchia, C

2017-04-15

The main aim of this paper was to assess the impact of Gluten-Friendly™ (GF) technology (Italian priority patent n° 102015000084813 filed on 17th December 2015) on wheat kernel endosperm morphology and gluten protein structure, using SEM, light and immunofluorescent microscopy. Microscopy was combined with immunodetection with specific antibodies for gliadins, γ-gliadins, LMW subunits and antigenic epitopes to gain a better understanding of the technology at a molecular level. The results showed significant changes to gluten proteins after GF treatment; cross-reactivity towards the antibodies recognizing almost the entire range of gluten proteins as well as the antigenic epitopes through the sequences QQSF, QQSY, PEQPFPQGC and QQPFP was significantly reduced. The present study confirms the results from our previous work and shows, for the first time, the mechanism by which a chemical-physical treatment abolishes the antigenic capacity of gluten. Copyright © 2016 Elsevier Ltd. All rights reserved.

SPED light sheet microscopy: fast mapping of biological system structure and function

PubMed Central

Tomer, Raju; Lovett-Barron, Matthew; Kauvar, Isaac; Andalman, Aaron; Burns, Vanessa M.; Sankaran, Sethuraman; Grosenick, Logan; Broxton, Michael; Yang, Samuel; Deisseroth, Karl

2016-01-01

The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light-sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca2+ imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function. PMID:26687363

Light Emitting Diode Flashlights as Effective and Inexpensive Light Sources for Fluorescence Microscopy

PubMed Central

Robertson, J. Brian; Zhang, Yunfei; Johnson, Carl Hirschie

2009-01-01

Summary Light-emitting diodes (LEDs) are becoming more commonly used as light sources for fluorescence microscopy. We describe the adaptation of a commercially available LED flashlight for use as a source for fluorescence excitation. This light source is long-lived, inexpensive, and is effective for excitation in the range of 440–600 nm. PMID:19772530

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Quantitative phase imaging of biological cells and tissues using singleshot white light interference microscopy and phase subtraction method for extended range of measurement

NASA Astrophysics Data System (ADS)

Mehta, Dalip Singh; Sharma, Anuradha; Dubey, Vishesh; Singh, Veena; Ahmad, Azeem

2016-03-01

We present a single-shot white light interference microscopy for the quantitative phase imaging (QPI) of biological cells and tissues. A common path white light interference microscope is developed and colorful white light interferogram is recorded by three-chip color CCD camera. The recorded white light interferogram is decomposed into the red, green and blue color wavelength component interferograms and processed it to find out the RI for different color wavelengths. The decomposed interferograms are analyzed using local model fitting (LMF)" algorithm developed for reconstructing the phase map from single interferogram. LMF is slightly off-axis interferometric QPI method which is a single-shot method that employs only a single image, so it is fast and accurate. The present method is very useful for dynamic process where path-length changes at millisecond level. From the single interferogram a wavelength-dependent quantitative phase imaging of human red blood cells (RBCs) are reconstructed and refractive index is determined. The LMF algorithm is simple to implement and is efficient in computation. The results are compared with the conventional phase shifting interferometry and Hilbert transform techniques.

Organic petrology of selected oil shale samples from lower Carboniferous Albert Formation, New Brunswick, Canada

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalkreuth, W.; Macauley, G.

1984-04-01

Incident light microscopy was used to describe maturation and composition of organic material in oil shale samples from the Lower Carboniferous Albert Formation of New Brunswick. The maturation level was determined in normal (white) light by measuring vitrinite reflectance and in fluorescent light by measuring fluorescence spectral of alginite B. Results indicate low to intermediate maturation for all of the samples. Composition was determined by maceral analysis. Alginite B is the major organic component in all samples having significant oil potential. Oil yields obtained from the Fischer Assay process, and oil and gas potentials from Rock-Eval analyses correlate to themore » amounts of alginite B and bituminite determined in the samples. In some of the samples characterized by similar high concentrations of alginite B, decrease in Fischer Assay yields and oil and gas potentials is related to an increase in maturation, as expected by increase in the fluorescence parameter lambda/sub max/ and red/green quotient of alginite B. Incident light microscopy, particularly with fluorescent light, offers a valuable tool for the identification of the organic matter in oil shales and for the evaluation of their oil and gas potentials.« less

Changes in surface morphology and mineralization level of human enamel following in-office bleaching with 35% hydrogen peroxide and light irradiation.

PubMed

Berger, Sandrine Bittencourt; Cavalli, Vanessa; Ambrosano, Glaucia Maria Bovi; Giannini, Marcelo

2010-01-01

The objective of this study was to evaluate the alterations on surface morphology and mineral loss of human enamel following in-office bleaching with 35% hydrogen peroxide and light irradiation. Dental enamel samples were obtained from human third molars and randomly divided into 10 groups (n = 10). The control group remained untreated. Bleached groups were treated with one of three whitening products. Bleaching was performed in a single session, during which bleaching gel was applied to the enamel surface three times for 10 minutes each time. During treatment, the bleaching agents were either irradiated by a halogen light or an LED/diode laser or were not irradiated at all. Microhardness testing was performed with a Knoop indentor and the surface morphologic observations were carried out by scanning electron microscopy (SEM). Cross-sectional microhardness (CSMH) and polarized light microscopy (PLM) were used to measure the depth of demineralization. The results revealed a significant decrease in surface microhardness values and changes to the enamel morphology after bleaching. CSMH and PLM showed that bleached enamel presented lower volume percentage of mineral up to 40 micrometers from the enamel surface and demineralization areas located in the subsuperficial region of enamel, respectively. It was concluded that 35% hydrogen peroxide can alter the surface morphology and the mineralization level of the dental enamel surface and sub-surface regardless of what type of bleaching light is used.

Microscopy and Image Analysis.

PubMed

McNamara, George; Difilippantonio, Michael; Ried, Thomas; Bieber, Frederick R

2017-07-11

This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Value of Reflected Light Microscopy in Teaching.

ERIC Educational Resources Information Center

Pasteris, Jill Dill

1983-01-01

Briefly reviews some optical and other physical properties of minerals that can be determined in reflected/incident light. Topics include optical properties of minerals, reflectance, internal reflections, color, bireflectance and reflection pleochroism, anisotropism, zonation, and reflected light microscopy as a teaching tool in undergraduate…

Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p.

PubMed

Bodvard, Kristofer; Wrangborg, David; Tapani, Sofia; Logg, Katarina; Sliwa, Piotr; Blomberg, Anders; Kvarnström, Mats; Käll, Mikael

2011-02-01

Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters. We here investigate how continuous illumination with blue light, corresponding to GFP excitation wavelengths, affects the localization pattern of Msn2p-GFP in budding yeast. The localization pattern was analyzed using a novel approach that combines wavelet decomposition and change point analysis. It was found that the Msn2p nucleocytoplasmic localization trajectories for individual cells exhibit up to three distinct and successive states; i) Msn2p localizes to the cytoplasm; ii) Msn2p rapidly shuttles between the cytoplasm and the nucleus; iii) Msn2p localizes to the nucleus. Many cells pass through all states consecutively at high light intensities, while at lower light intensities most cells only reach states i) or ii). This behaviour strongly indicates that continuous light exposure gradually increases the stress level over time, presumably through continuous accumulation of toxic photoproducts, thereby forcing the cell through a bistable region corresponding to nucleocytoplasmic oscillations. We also show that the localization patterns are dependent on protein kinase A (PKA) activity, i.e. yeast cells with constantly low PKA activity showed a stronger stress response. In particular, the nucleocytoplasmic oscillation frequency was found to be significantly higher for cells with low PKA activity for all light intensities. 2010 Elsevier B.V. All rights reserved.

Hyperspectral microscopy to identify foodborne bacteria with optimum lighting source

USDA-ARS?s Scientific Manuscript database

Hyperspectral microscopy is an emerging technology for rapid detection of foodborne pathogenic bacteria. Since scattering spectral signatures from hyperspectral microscopic images (HMI) vary with lighting sources, it is important to select optimal lights. The objective of this study is to compare t...

Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

PubMed Central

Zhao, Ming; Zhang, Han; Li, Yu; Ashok, Amit; Liang, Rongguang; Zhou, Weibin; Peng, Leilei

2014-01-01

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM. PMID:24876996

Full optical model of micro-endoscope with optical coherence microscopy, multiphoton microscopy and visible capabilities

NASA Astrophysics Data System (ADS)

Vega, David; Kiekens, Kelli C.; Syson, Nikolas C.; Romano, Gabriella; Baker, Tressa; Barton, Jennifer K.

2018-02-01

While Optical Coherence Microscopy (OCM), Multiphoton Microscopy (MPM), and narrowband imaging are powerful imaging techniques that can be used to detect cancer, each imaging technique has limitations when used by itself. Combining them into an endoscope to work in synergy can help achieve high sensitivity and specificity for diagnosis at the point of care. Such complex endoscopes have an elevated risk of failure, and performing proper modelling ensures functionality and minimizes risk. We present full 2D and 3D models of a multimodality optical micro-endoscope to provide real-time detection of carcinomas, called a salpingoscope. The models evaluate the endoscope illumination and light collection capabilities of various modalities. The design features two optical paths with different numerical apertures (NA) through a single lens system with a scanning optical fiber. The dual path is achieved using dichroic coatings embedded in a triplet. A high NA optical path is designed to perform OCM and MPM while a low NA optical path is designed for the visible spectrum to navigate the endoscope to areas of interest and narrowband imaging. Different tests such as the reflectance profile of homogeneous epithelial tissue were performed to adjust the models properly. Light collection models for the different modalities were created and tested for efficiency. While it is challenging to evaluate the efficiency of multimodality endoscopes, the models ensure that the system is design for the expected light collection levels to provide detectable signal to work for the intended imaging.

Polarized light and scanning electron microscopic investigation of enamel hypoplasia in primary teeth.

PubMed

Sabel, Nina; Klingberg, Gunilla; Dietz, Wolfram; Nietzsche, Sandor; Norén, Jörgen G

2010-01-01

Enamel hypoplasia is a developmental disturbance during enamel formation, defined as a macroscopic defect in the enamel, with a reduction of the enamel thickness with rounded, smooth borders. Information on the microstructural level is still limited, therefore further studies are of importance to better understand the mechanisms behind enamel hypoplasia. To study enamel hypoplasia in primary teeth by means of polarized light microscopy and scanning electron microscopy. Nineteen primary teeth with enamel hypoplasia were examined in a polarized light microscope and in a scanning electron microscope. The cervical and incisal borders of the enamel hypoplasia had a rounded appearance, as the prisms in the rounded cervical area of the hypoplasia were bent. The rounded borders had a normal surface structure whereas the base of the defects appeared rough and porous. Morphological findings in this study indicate that the aetiological factor has a short duration and affects only certain ameloblasts. The bottom of the enamel hypoplasia is porous and constitutes possible pathways for bacteria into the dentin.

A Simplified, Low-Cost Method for Polarized Light Microscopy

PubMed Central

Maude, Richard J.; Buapetch, Wanchana; Silamut, Kamolrat

2009-01-01

Malaria pigment is an intracellular inclusion body that appears in blood and tissue specimens on microscopic examination and can help in establishing the diagnosis of malaria. In simple light microscopy, it can be difficult to discern from cellular background and artifacts. It has long been known that if polarized light microscopy is used, malaria pigment can be much easier to distinguish. However, this technique is rarely used because of the need for a relatively costly polarization microscope. We describe a simple and economical technique to convert any standard light microscope suitable for examination of malaria films into a polarization microscope. PMID:19861611

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

A Comparison of Honey Bee-Collected Pollen From Working Agricultural Lands Using Light Microscopy and ITS Metabarcoding.

PubMed

Smart, M D; Cornman, R S; Iwanowicz, D D; McDermott-Kubeczko, M; Pettis, J S; Spivak, M S; Otto, C R V

2017-02-01

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010-2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

Reduced Autophagy in 5-Fluorouracil Resistant Colon Cancer Cells

PubMed Central

Yao, Cheng Wen; Kang, Kyoung Ah; Piao, Mei Jing; Ryu, Yea Seong; Fernando, Pattage Madushan Dilhara Jayatissa; Oh, Min Chang; Park, Jeong Eon; Shilnikova, Kristina; Na, Soo-Young; Jeong, Seung Uk; Boo, Sun-Jin; Hyun, Jin Won

2017-01-01

We investigated the role of autophagy in SNUC5/5-FUR, 5-fluorouracil (5-FU) resistant SNUC5 colon cancer cells. SNUC5/5-FUR cells exhibited low level of autophagy, as determined by light microscopy, confocal microscopy, and flow cytometry following acridine orange staining, and the decreased level of GFP-LC3 puncta. In addition, expression of critical autophagic proteins such as Atg5, Beclin-1 and LC3-II and autophagic flux was diminished in SNUC5/5-FUR cells. Whereas production of reactive oxygen species (ROS) was significantly elevated in SNUC5/5-FUR cells, treatment with the ROS inhibitor N-acetyl cysteine further reduced the level of autophagy. Taken together, these results indicate that decreased autophagy is linked to 5-FU resistance in SNUC5 colon cancer cells. PMID:27737524

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

PubMed Central

Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem

2017-01-01

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level. PMID:28426025

Light Microscopy's New Jobs

NASA Astrophysics Data System (ADS)

Ritsch-Marte, Monika

2009-04-01

300 years since the first glimpse through the earliest microscopes, light microscopy is still an active field of research, breaking new frontiers in optical imaging and even becoming a means of mechanical manipulation of microparticles.

Quantitative Assessment of Fat Levels in Caenorhabditis elegans Using Dark Field Microscopy

PubMed Central

Fouad, Anthony D.; Pu, Shelley H.; Teng, Shelly; Mark, Julian R.; Fu, Moyu; Zhang, Kevin; Huang, Jonathan; Raizen, David M.; Fang-Yen, Christopher

2017-01-01

The roundworm Caenorhabditis elegans is widely used as a model for studying conserved pathways for fat storage, aging, and metabolism. The most broadly used methods for imaging fat in C. elegans require fixing and staining the animal. Here, we show that dark field images acquired through an ordinary light microscope can be used to estimate fat levels in worms. We define a metric based on the amount of light scattered per area, and show that this light scattering metric is strongly correlated with worm fat levels as measured by Oil Red O (ORO) staining across a wide variety of genetic backgrounds and feeding conditions. Dark field imaging requires no exogenous agents or chemical fixation, making it compatible with live worm imaging. Using our method, we track fat storage with high temporal resolution in developing larvae, and show that fat storage in the intestine increases in at least one burst during development. PMID:28404661

Controlled power delivery for super-resolution imaging of biological samples using digital micromirror device

NASA Astrophysics Data System (ADS)

Valiya Peedikakkal, Liyana; Cadby, Ashley

2017-02-01

Localization based super resolution images of a biological sample is generally achieved by using high power laser illumination with long exposure time which unfortunately increases photo-toxicity of a sample, making super resolution microscopy, in general, incompatible with live cell imaging. Furthermore, the limitation of photobleaching reduces the ability to acquire time lapse images of live biological cells using fluorescence microscopy. Digital Light Processing (DLP) technology can deliver light at grey scale levels by flickering digital micromirrors at around 290 Hz enabling highly controlled power delivery to samples. In this work, Digital Micromirror Device (DMD) is implemented in an inverse Schiefspiegler telescope setup to control the power and pattern of illumination for super resolution microscopy. We can achieve spatial and temporal patterning of illumination by controlling the DMD pixel by pixel. The DMD allows us to control the power and spatial extent of the laser illumination. We have used this to show that we can reduce the power delivered to the sample to allow for longer time imaging in one area while achieving sub-diffraction STORM imaging in another using higher power densities.

Malaria Diagnosis Using a Mobile Phone Polarized Microscope

NASA Astrophysics Data System (ADS)

Pirnstill, Casey W.; Coté, Gerard L.

2015-08-01

Malaria remains a major global health burden, and new methods for low-cost, high-sensitivity, diagnosis are essential, particularly in remote areas with low-resource around the world. In this paper, a cost effective, optical cell-phone based transmission polarized light microscope system is presented for imaging the malaria pigment known as hemozoin. It can be difficult to determine the presence of the pigment from background and other artifacts, even for skilled microscopy technicians. The pigment is much easier to observe using polarized light microscopy. However, implementation of polarized light microscopy lacks widespread adoption because the existing commercial devices have complicated designs, require sophisticated maintenance, tend to be bulky, can be expensive, and would require re-training for existing microscopy technicians. To this end, a high fidelity and high optical resolution cell-phone based polarized light microscopy system is presented which is comparable to larger bench-top polarized microscopy systems but at much lower cost and complexity. The detection of malaria in fixed and stained blood smears is presented using both, a conventional polarized microscope and our cell-phone based system. The cell-phone based polarimetric microscopy design shows the potential to have both the resolution and specificity to detect malaria in a low-cost, easy-to-use, modular platform.

Malaria Diagnosis Using a Mobile Phone Polarized Microscope

PubMed Central

Pirnstill, Casey W.; Coté, Gerard L.

2015-01-01

Malaria remains a major global health burden, and new methods for low-cost, high-sensitivity, diagnosis are essential, particularly in remote areas with low-resource around the world. In this paper, a cost effective, optical cell-phone based transmission polarized light microscope system is presented for imaging the malaria pigment known as hemozoin. It can be difficult to determine the presence of the pigment from background and other artifacts, even for skilled microscopy technicians. The pigment is much easier to observe using polarized light microscopy. However, implementation of polarized light microscopy lacks widespread adoption because the existing commercial devices have complicated designs, require sophisticated maintenance, tend to be bulky, can be expensive, and would require re-training for existing microscopy technicians. To this end, a high fidelity and high optical resolution cell-phone based polarized light microscopy system is presented which is comparable to larger bench-top polarized microscopy systems but at much lower cost and complexity. The detection of malaria in fixed and stained blood smears is presented using both, a conventional polarized microscope and our cell-phone based system. The cell-phone based polarimetric microscopy design shows the potential to have both the resolution and specificity to detect malaria in a low-cost, easy-to-use, modular platform. PMID:26303238

Further description of Cruzia tentaculata (Rudolphi, 1819) Travassos, 1917 (Nematoda: Cruzidae) by light and scanning electron microscopy.

PubMed

Adnet, F A O; Anjos, D H S; Menezes-Oliveira, A; Lanfredi, R M

2009-04-01

Species of Cruzia are parasites of the large intestine of marsupials, reptiles, amphibians, and mammalians. Cruzia tentaculata specimens were collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Colombia (new geographical record) and from Brazil and analyzed by light and scanning electron microscopy. The morphology of males and females by light microscopy corroborated most of the previous description and the ultrastructure by scanning electron microscopy evidence: the topography of the cuticle, deirids, amphids, phasmids in both sexes, a pair of papillae near the vulva opening, and the number and location of male caudal papillae, adding new features for species identification only observed by this technique.

White Light–Emitting Diodes (LEDs) at Domestic Lighting Levels and Retinal Injury in a Rat Model

PubMed Central

Shang, Yu-Man; Wang, Gen-Shuh; Sliney, David; Lee, Li-Ling

2013-01-01

Background: Light-emitting diodes (LEDs) deliver higher levels of blue light to the retina than do conventional domestic light sources. Chronic exposure to high-intensity light (2,000–10,000 lux) has previously been found to result in light-induced retinal injury, but chronic exposure to relatively low-intensity (750 lux) light has not been previously assessed with LEDs in a rodent model. Objective: We examined LED-induced retinal neuronal cell damage in the Sprague-Dawley rat using functional, histological, and biochemical measurements. Methods: We used blue LEDs (460 nm) and full-spectrum white LEDs, coupled with matching compact fluorescent lights, for exposures. Pathological examinations included electroretinogram, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM). We also measured free radical production in the retina to determine the oxidative stress level. Results: H&E staining and TEM revealed apoptosis and necrosis of photoreceptors, which indicated blue-light induced photochemical injury of the retina. Free radical production in the retina was increased in LED-exposed groups. IHC staining demonstrated that oxidative stress was associated with retinal injury. Although we found serious retinal light injury in LED groups, the compact fluorescent lamp (CFL) groups showed moderate to mild injury. Conclusion: Our results raise questions about adverse effects on the retina from chronic exposure to LED light compared with other light sources that have less blue light. Thus, we suggest a precautionary approach with regard to the use of blue-rich “white” LEDs for general lighting. Citation: Shang YM, Wang GS, Sliney D, Yang CH, Lee LL. 2014. White light–emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ Health Perspect 122:269–276; http://dx.doi.org/10.1289/ehp.1307294 PMID:24362357

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching

PubMed Central

Gerbich, Therese M.; Rana, Kishan; Suzuki, Aussie; Schaefer, Kristina N.; Heppert, Jennifer K.; Boothby, Thomas C.; Allbritton, Nancy L.; Gladfelter, Amy S.; Maddox, Amy S.

2018-01-01

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution. PMID:29490939

Retracing in correlative light electron microscopy: where is my object of interest?

PubMed

Hodgson, Lorna; Nam, David; Mantell, Judith; Achim, Alin; Verkade, Paul

2014-01-01

Correlative light electron microscopy (CLEM) combines the strengths of light and electron microscopy in a single experiment. There are many ways to perform a CLEM experiment and a variety of microscopy modalities can be combined either on separate instruments or as completely integrated solutions. In general, however, a CLEM experiment can be divided into three parts: probes, processing, and analysis. Most of the existing technologies are focussed around the development and use of probes or describe processing methodologies that explain or circumvent some of the compromises that need to be made when performing both light and electron microscopy on the same sample. So far, relatively little attention has been paid to the analysis part of CLEM experiments. Although it is an essential part of each CLEM experiment, it is usually a cumbersome manual process. Here, we briefly discuss each of the three above-mentioned steps, with a focus on the analysis part. We will also introduce an automated registration algorithm that can be applied to the analysis stage to enable the accurate registration of LM and EM images. This facilitates tracing back the right cell/object seen in the light microscope in the EM. © 2014 Elsevier Inc. All rights reserved.

Multilayer mounting for long-term light sheet microscopy of zebrafish.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-02-27

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

Correlative light-electron fractography for fatigue striations characterization in metallic alloys.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-09-01

The correlative light-electron fractography technique combines correlative microscopy concepts to the extended depth-from-focus reconstruction method, associating the reliable topographic information of 3-D maps from light microscopy ordered Z-stacks to the finest lateral resolution and large focus depth from scanning electron microscopy. Fatigue striations spacing analysis can be precisely measured, by correcting the mean surface tilting with the knowledge of local elevation data from elevation maps. This new technique aims to improve the accuracy of quantitative fractography in fatigue fracture investigations. Copyright © 2013 Wiley Periodicals, Inc.

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

PubMed Central

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology. PMID:24637614

Super-resolution optical microscopy for studying membrane structure and dynamics.

PubMed

Sezgin, Erdinc

2017-07-12

Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

eduSPIM: Light Sheet Microscopy in the Museum

PubMed Central

Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light Sheet Microscopy in the Museum Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. Design Principles of an Educational Light Sheet Microscope To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The eduSPIM Design Is Tailored Easily to Fit Numerous Applications The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided. PMID:27560188

Applications of microscopy in Salmonella research.

PubMed

Malt, Layla M; Perrett, Charlotte A; Humphrey, Suzanne; Jepson, Mark A

2015-01-01

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Statistical parametric mapping of stimuli-evoked changes in quantitative blood flow using extended-focus optical coherence microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Marchand, Paul J.; Bouwens, Arno; Shamaei, Vincent; Nguyen, David; Extermann, Jerome; Bolmont, Tristan; Lasser, Theo

2016-03-01

Magnetic Resonance Imaging has revolutionised our understanding of brain function through its ability to image human cerebral structures non-invasively over the entire brain. By exploiting the different magnetic properties of oxygenated and deoxygenated blood, functional MRI can indirectly map areas undergoing neural activation. Alongside the development of fMRI, powerful statistical tools have been developed in an effort to shed light on the neural pathways involved in processing of sensory and cognitive information. In spite of the major improvements made in fMRI technology, the obtained spatial resolution of hundreds of microns prevents MRI in resolving and monitoring processes occurring at the cellular level. In this regard, Optical Coherence Microscopy is an ideal instrumentation as it can image at high spatio-temporal resolution. Moreover, by measuring the mean and the width of the Doppler spectra of light scattered by moving particles, OCM allows extracting the axial and lateral velocity components of red blood cells. The ability to assess quantitatively total blood velocity, as opposed to classical axial velocity Doppler OCM, is of paramount importance in brain imaging as a large proportion of cortical vascular is oriented perpendicularly to the optical axis. We combine here quantitative blood flow imaging with extended-focus Optical Coherence Microscopy and Statistical Parametric Mapping tools to generate maps of stimuli-evoked cortical hemodynamics at the capillary level.

Even illumination in total internal reflection fluorescence microscopy using laser light.

PubMed

Fiolka, R; Belyaev, Y; Ewers, H; Stemmer, A

2008-01-01

In modern fluorescence microscopy, lasers are a widely used source of light, both for imaging in total internal reflection and epi-illumination modes. In wide-field imaging, scattering of highly coherent laser light due to imperfections in the light path typically leads to nonuniform illumination of the specimen, compromising image analysis. We report the design and construction of an objective-launch total internal reflection fluorescence microscopy system with excellent evenness of specimen illumination achieved by azimuthal rotation of the incoming illuminating laser beam. The system allows quick and precise changes of the incidence angle of the laser beam and thus can also be used in an epifluorescence mode. 2007 Wiley-Liss, Inc

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

PubMed

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

A Photomicrography Primer.

ERIC Educational Resources Information Center

Davidson, Michael W.

1991-01-01

Describes techniques and equipment which allows school microscopes to perform crossed-polarized light microscopy, reflected light microscopy, and photomicrography. Provides information on using chemicals from a high school stockroom to view crystals, viewing integrated circuits, and capturing images on film. Lists possible independent student…

A Comparative Study Between Smartphone-Based Microscopy and Conventional Light Microscopy in 1021 Dermatopathology Specimens.

PubMed

Jahan-Tigh, Richard R; Chinn, Garrett M; Rapini, Ronald P

2016-01-01

The incorporation of high-resolution cameras into smartphones has allowed for a variety of medical applications including the use of lens attachments that provide telescopic, macroscopic, and dermatoscopic data, but the feasibility and performance characteristics of such a platform for use in dermatopathology have not been described. To determine the diagnostic performance of a smartphone microscope compared to traditional light microscopy in dermatopathology specimens. A simple smartphone microscope constructed with a 3-mm ball lens was used to prospectively evaluate 1021 consecutive dermatopathology cases in a blinded fashion. Referred, consecutive specimens from the community were evaluated at a single university hospital. The performance characteristics of the smartphone platform were calculated by using conventional light microscopy as the gold standard. The sensitivity and specificity for the diagnosis of melanoma, nonmelanoma skin cancers, and other miscellaneous conditions by the phone microscopy platform, as compared with traditional light microscopy, were calculated. For basal cell carcinoma (n = 136), the sensitivity and specificity of smartphone microscopy were 95.6% and 98.1%, respectively. The sensitivity and specificity for squamous cell carcinoma (n = 94) were 89.4% and 97.3%, respectively. The lowest sensitivity was found in melanoma (n = 15) at 60%, although the specificity was high at 99.1%. The accuracy of diagnosis of inflammatory conditions and other neoplasms was variable. Mobile phone-based microscopy has excellent performance characteristics for the inexpensive diagnosis of nonmelanoma skin cancers in a setting where a traditional microscope is not available.

Super-resolution and super-localization microscopy: A novel tool for imaging chemical and biological processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Bin

2015-01-01

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the singlemore » molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).« less

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy

PubMed Central

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM). PMID:29568263

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

PubMed

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

PubMed

Pluk, H; Stokes, D J; Lich, B; Wieringa, B; Fransen, J

2009-03-01

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Video image processing greatly enhances contrast, quality, and speed in polarization-based microscopy

PubMed Central

1981-01-01

Video cameras with contrast and black level controls can yield polarized light and differential interference contrast microscope images with unprecedented image quality, resolution, and recording speed. The theoretical basis and practical aspects of video polarization and differential interference contrast microscopy are discussed and several applications in cell biology are illustrated. These include: birefringence of cortical structures and beating cilia in Stentor, birefringence of rotating flagella on a single bacterium, growth and morphogenesis of echinoderm skeletal spicules in culture, ciliary and electrical activity in a balancing organ of a nudibranch snail, and acrosomal reaction in activated sperm. PMID:6788777

Light scattering and light transmittance of cadaver eye-explanted intraocular lenses of different materials.

PubMed

Morris, Caleb; Werner, Liliana; Barra, Daniel; Liu, Erica; Stallings, Shannon; Floyd, Anne

2014-01-01

To evaluate light scattering and light transmittance in cadaver eye-explanted intraocular lenses (IOLs) manufactured from different materials. John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Experimental study. Forty-nine pseudophakic cadaver eyes were selected according to IOL material/type and implantation duration, and the IOLs were explanted. Hydrophobic acrylic, hydrophilic acrylic, poly(methyl methacrylate) (PMMA), and silicone IOLs were included. Gross and light microscopy was performed for all IOLs. Light scattering was measured with an EAS 1000 Scheimpflug camera, and light transmittance was assessed using a Lambda 35 UV/Vis spectrophotometer (single-beam configuration with an RSA PE-20 integrating sphere). Analyses were performed at room temperature in the hydrated state and compared with analyses of controls. The highest levels of surface light scattering were measured for 3-piece hydrophobic acrylic, which was also the IOL type with the longest implantation duration among the Acrysof hydrophobic acrylic IOLs. Hydrophilic acrylic, PMMA, and silicone IOLs exhibited relatively low light-scattering levels. The lowest light-scattering levels were observed with PMMA IOLs (1-piece looped and 3-piece) and plate silicone IOLs, which represent the IOL types with the longest implantation duration in this series. Light transmittance values measured for all IOL types appeared to be similar to the values of the corresponding control IOLs. The phenomenon of surface light scattering (nanoglistenings) is more particularly related to hydrophobic acrylic IOLs and increases with implantation time. No significant effect of surface light scattering on IOL light transmittance was found. Copyright © 2013 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Daunton, N. G.

1992-01-01

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Green light for quantitative live-cell imaging in plants.

PubMed

Grossmann, Guido; Krebs, Melanie; Maizel, Alexis; Stahl, Yvonne; Vermeer, Joop E M; Ott, Thomas

2018-01-29

Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number of methodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants. © 2018. Published by The Company of Biologists Ltd.

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.

Summary of 2016 Light Microscopy Module (LMM) Physical Science Experiments on ISS. Update of LMM Science Experiments and Facility Capabilities

NASA Technical Reports Server (NTRS)

Sicker, Ronald J.; Meyer, William V.; Foster, William M.; Fletcher, William A.; Williams, Stuart J.; Lee, Chang-Soo

2016-01-01

This presentation will feature a series of short, entertaining, and informative videos that describe the current status and science support for the Light Microscopy Module (LMM) facility on the International Space Station. These interviews will focus on current experiments and provide an overview of future capabilities. The recently completed experiments include nano-particle haloing, 3-D self-assembly with Janus particles and a model system for nano-particle drug delivery. The videos will share perspectives from the scientists, engineers, and managers working with the NASA Light Microscopy program.

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution.

PubMed

Coates, Colin G; Denvir, Donal J; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

2004-01-01

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD. Copyright 2004 Society of Photo-Optical Instrumentation Engineers.

Recent advancements in structured-illumination microscopy toward live-cell imaging.

PubMed

Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

2015-08-01

Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

PubMed

Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

2015-10-01

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

Correlative light and electron microscopic detection of GFP-labeled proteins using modular APEX.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Parton, Robert G

2017-01-01

The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy

NASA Astrophysics Data System (ADS)

Baek, Jongho; Lou, Jieqiong; Coelho, Simao; Lim, Yean Jin; Seidlitz, Silvia; Nicovich, Philip R.; Wunder, Christian; Johannes, Ludger; Gaus, Katharina

2017-04-01

Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

Longitudinal spatial coherence gated high-resolution tomography and quantitative phase microscopy of biological cells and tissues with uniform illumination

NASA Astrophysics Data System (ADS)

Mehta, Dalip Singh; Ahmad, Azeem; Dubey, Vishesh; Singh, Veena; Butola, Ankit; Mohanty, Tonmoy; Nandi, Sreyankar

2018-02-01

We report longitudinal spatial coherence (LSC) gated high-resolution tomography and quantitative phase microscopy of biological cells and tissues with uniform illumination using laser as a light source. To accomplish this a pseudo thermal light source was synthesized by passing laser beams through an optical system, which is basically a speckle reduction system with combined effect of spatial, temporal, angular and polarisation diversity. The longitudinal spatial coherence length of such light was significantly reduced by synthesizing a pseudo thermal source with the combined effect of spatial, angular and temporal diversity. This results in a low spatially coherent (i.e., broad angular frequency spectrum) light source with narrow temporal frequency spectrum. Light from such a pseudo thermal light source was passed through an interference microscope with varying magnification, such as, 10X and 50X. The interference microscope was used for full-field OCT imaging of multilayer objects and topography of industrial objects. Experimental results of optical sectioning of multilayer biological objects with high axial-resolution less than 10μm was achieved which is comparable to broadband white light source. The synthesized light source with reduced speckles having uniform illumination on the sample, which can be very useful for fluorescence microscopy as well as quantitative phase microscopy with less phase noise. The present system does not require any dispersion compensation optical system for biological samples as a highly monochromatic light source is used.

Optical Diagnostics in Medicine

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor

2003-03-01

Light has a unique potential for non-invasive tissue diagnosis. The relatively short wavelength of light allows imaging of tissue at the resolution of histopathology. While strong multiple scattering of light in tissue makes attainment of this resolution difficult for thick tissues, most pathology emanates from epithelial surfaces. Therefore, high-resolution diagnosis of many important diseases may be achieved by transmitting light to the surface of interest. The recent fiber-optic implementation of technologies that reject multiple scattering, such as confocal microscopy and optical low coherence interferometry, have brought us one step closer to realizing non-invasive imaging of architectural and cellular features of tissue. Optical coherence tomography (OCT) can produce high-resolution cross-sectional images of biological structures. Clinical OCT studies conducted in the gastrointestinal tract and cardiovascular system have shown that OCT is capable of providing images of the architectural (> 20 µm) microanatomy of a variety of epithelial tissues, including the layered structure of squamous epithelium and arterial vessels. Fine Needle Aspiration- Low Coherence Interferometry (FNA-LCI) is another optical diagnostics technique, which is a suitable solution to increase the effectiveness of the FNA procedures. LCI is capable of measuring depth resolved (axial, z) tissue structure, birefringence, flow (Doppler shift), and spectra at a resolution of several microns. Since LCI systems are fiber-optic based, LCI probes may easily fit within the bore of a fine gauge needle, allowing diagnostic information to be obtained directly from the FNA biopsy site. Fiber optic spectrally encoded confocal microscopy (SECM) is a new confocal microscopy method, which eliminates the need for rapid beam scanning within the optical probe. This advance enables confocal microscopy to be performed through small diameter probes and will allow assessment of internal human tissues in vivo at the cellular level. A detailed description of several fiber optics based systems for early diseases diagnosis, as well as preliminary clinic results, will be presented.

Evaluation of the Surface Characteristics of Various Implant Abutment Materials Using Confocal Microscopy and White Light Interferometry.

PubMed

Park, Jun-Beom; Yang, Seung-Min; Ko, Youngkyung

2015-12-01

The purpose of this study was to evaluate the surface characteristics of various implant abutment materials, such as of titanium alloy (Ti6Al4V; Ma), machined cobalt-chrome-molybdenum alloy (CCM), titanium nitride coating on a titanium alloy disc (TiN), anodic oxidized titanium alloy disc (AO), composite resin coating on a titanium alloy disc (Res), and zirconia disc (Zr), using confocal microscopy and white light interferometry. Measurements from the 2 methods were evaluated to see if these methods would give equivalent results. The precision of measurements were evaluated by the coefficient of variation. Five discs each of Ma, CCM, TiN, AO, Res, and Zr were used. The surface roughness was evaluated by confocal laser microscopy and white light interferometry. Confocal microscopy showed that the Res group showed significantly greater Ra, Rq, Rz, Sa, Sq, and Sz values compared with those of the Ma group (P < 0.05). The white light interferometry results showed that the Res group had significantly higher Ra, Rq, Rz, Rt, Sa, Sq, Sz, and Sdr values compared with the Ma group (P < 0.05). All the roughness parameters obtained from the 2 methods differed, and the Sa values of the Zr group from confocal microscopy were greater by 0.163 μm than those obtained by white light interferometry. Least difference was seen in the TiN group where the difference was 0.058 μm. Roughness parameters of different abutment materials varied significantly. Precision of measurement differed according to the characteristics of the material used. White light interferometry could be recommended for measurement of TiN and AO. Confocal microscopy gave more precise measurements for Ma and CCM groups. The optical characteristics of the surface should be considered before choosing the examination method.

Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy1[OPEN

PubMed Central

Cavonius, Lillie; Fink, Helen; Kiskis, Juris; Albers, Eva; Undeland, Ingrid; Enejder, Annika

2015-01-01

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r2 = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation. PMID:25583924

ALA-PDT elicits oxidative damage and apoptosis in UVB-induced premature senescence of human skin fibroblasts.

PubMed

Zhou, Bing-Rong; Zhang, Li-Chao; Permatasari, Felicia; Liu, Juan; Xu, Yang; Luo, Dan

2016-06-01

5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has been used for the treatment of skin photoaging. It can significantly improve the appearance of fine lines, dotted pigmentation, and roughness of photoaged skin. However, the mechanisms by which ALA-PDT yields rejuvenating effects on photoaged skin have not been well elucidated. Thus, in this study we explored the effects of ALA-PDT in photoaged fibroblasts. We established a stress-induced premature senescence (SIPS) model by repeated exposures of human dermal fibroblasts (HDFs) to ultraviolet B (UVB) irradiation. Cells were irradiated by red light laser at 635nm wavelength (50mW/cm(2)). Intracellular protoporphyrin IX (PpIX) was detected by confocal microscopy. Intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) change were detected by fluorescence microscopy and flow cytometry. Morphological changes were observed by optical microscopy. Proliferative activity was measured by a cell counting kit-8 (CCK-8). Cell apoptosis was detected by fluorescence microscopy using Hoechst staining and flow cytometry using annexin V/propidium Iodide double staining. Intracellular PpIX fluorescence in UVB-induced premature senescent HDFs (UVB-SIPS-HDFs) reached the highest intensity after incubation with 1.00mmol/L ALA for 6h (P<0.05). Compared with control group, intracellular ROS level, MMP, and apoptotic rate were increased (P<0.05) and proliferative activity was decreased (P<0.05) in UVB-SIPS-HDFs treated with ALA-PDT, which were positively correlated to ALA incubation time and red light laser dose. Our study demonstrated that ALA-PDT elicits oxidative damage and apoptosis in photoaged fibroblasts in vitro, which may be the basis for the rejuvenating effects on photoaged skin. Copyright © 2016 Elsevier B.V. All rights reserved.

Low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy.

PubMed

Carlson, David B; Evans, James E

2011-06-05

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

PubMed

Johnson, Sam A

2015-01-01

Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches. © 2014 Wiley Periodicals, Inc.

Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy

PubMed Central

Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

2015-01-01

Purpose To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Methods Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Results Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Conclusion Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite. PMID:26445524

Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

2015-01-01

To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite.

Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy

PubMed Central

Cha, Jae Won; Ballesta, Jerome; So, Peter T.C.

2010-01-01

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration. PMID:20799824

Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy.

PubMed

Cha, Jae Won; Ballesta, Jerome; So, Peter T C

2010-01-01

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration.

Near-infrared branding efficiently correlates light and electron microscopy.

PubMed

Bishop, Derron; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

2011-06-05

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

Invited review article: Advanced light microscopy for biological space research.

PubMed

De Vos, Winnok H; Beghuin, Didier; Schwarz, Christian J; Jones, David B; van Loon, Jack J W A; Bereiter-Hahn, Juergen; Stelzer, Ernst H K

2014-10-01

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

Invited Review Article: Advanced light microscopy for biological space research

NASA Astrophysics Data System (ADS)

De Vos, Winnok H.; Beghuin, Didier; Schwarz, Christian J.; Jones, David B.; van Loon, Jack J. W. A.; Bereiter-Hahn, Juergen; Stelzer, Ernst H. K.

2014-10-01

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

[Cadmium citotoxicity in mice hepatocytes and implications on tropical environments].

PubMed

Marcano, Letty; Faría, Clarisa de R; Carruyo, Ingrid; Montiel, Xiomara

2006-06-01

We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

Silver stain for electron microscopy

NASA Technical Reports Server (NTRS)

Corbett, R. L.

1972-01-01

Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy.

Whole-animal imaging with high spatio-temporal resolution

NASA Astrophysics Data System (ADS)

Chhetri, Raghav; Amat, Fernando; Wan, Yinan; Höckendorf, Burkhard; Lemon, William C.; Keller, Philipp J.

2016-03-01

We developed isotropic multiview (IsoView) light-sheet microscopy in order to image fast cellular dynamics, such as cell movements in an entire developing embryo or neuronal activity throughput an entire brain or nervous system, with high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To achieve high temporal resolution and high spatial resolution at the same time, IsoView microscopy rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. In a post-processing step, these four views are then combined by means of high-throughput multiview deconvolution to yield images with a system resolution of ≤ 450 nm in all three dimensions. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 μm and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional (spatially anisotropic) light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, such as lattice lightsheet microscopy or diSPIM, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

Microwave Processing of Crowns from Winter Cereals for Light Microscopy.

USDA-ARS?s Scientific Manuscript database

Microwave processing of tissue considerably shortens the time it takes to prepare samples for light and electron microscopy. However, plant tissues from different species and different regions of the plant respond differently making it impossible to use a single protocol for all plant tissue. The ...

Orbital angular momentum light in microscopy

PubMed Central

2017-01-01

Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application. This article is part of the themed issue ‘Optical orbital angular momentum’. PMID:28069768

Wide-field imaging through scattering media by scattered light fluorescence microscopy

NASA Astrophysics Data System (ADS)

Zhou, Yulan; Li, Xun

2017-08-01

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

Condenser-free contrast methods for transmitted-light microscopy

PubMed Central

WEBB, K F

2015-01-01

Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared. PMID:25226859

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

PubMed

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

Three-dimensional wide-field pump-probe structured illumination microscopy

PubMed Central

Kim, Yang-Hyo; So, Peter T.C.

2017-01-01

We propose a new structured illumination scheme for achieving depth resolved wide-field pump-probe microscopy with sub-diffraction limit resolution. By acquiring coherent pump-probe images using a set of 3D structured light illumination patterns, a 3D super-resolution pump-probe image can be reconstructed. We derive the theoretical framework to describe the coherent image formation and reconstruction scheme for this structured illumination pump-probe imaging system and carry out numerical simulations to investigate its imaging performance. The results demonstrate a lateral resolution improvement by a factor of three and providing 0.5 µm level axial optical sectioning. PMID:28380860

Advanced Colloids Experiment (ACE) Science Overview

NASA Technical Reports Server (NTRS)

Meyer, William V.; Sicker, Ronald J.; Chiaramonte, Francis P.; Luna, Unique J.; Chaiken, Paul M.; Hollingsworth, Andrew; Secanna, Stefano; Weitz, David; Lu, Peter; Yodh, Arjun;

FIR Light Microscopy Module Set Up

NASA Image and Video Library

2009-11-09

ISS021-E-022460 (9 Nov. 2009) --- Canadian Space Agency astronaut Robert Thirsk, Expedition 21 flight engineer, installs the Light Microscopy Module (LMM) Spindle Bracket Assembly in the Fluids Integrated Rack (FIR) in the Destiny laboratory of the International Space Station. NASA astronaut Nicole Stott (out of frame), flight engineer, assisted Thirsk.

FIR Light Microscopy Module Set Up

NASA Image and Video Library

2009-11-09

ISS021-E-022459 (9 Nov. 2009) --- NASA astronaut Nicole Stott, Expedition 21 flight engineer, installs the Light Microscopy Module (LMM) Spindle Bracket Assembly in the Fluids Integrated Rack (FIR) in the Destiny laboratory of the International Space Station. Canadian Space Agency astronaut Robert Thirsk (out of frame) assisted Stott.

Correlative near-infrared light and cathodoluminescence microscopy using Y2O3:Ln, Yb (Ln = Tm, Er) nanophosphors for multiscale, multicolour bioimaging

PubMed Central

Fukushima, S.; Furukawa, T.; Niioka, H.; Ichimiya, M.; Sannomiya, T.; Tanaka, N.; Onoshima, D.; Yukawa, H.; Baba, Y.; Ashida, M.; Miyake, J.; Araki, T.; Hashimoto, M.

2016-01-01

This paper presents a new correlative bioimaging technique using Y2O3:Tm, Yb and Y2O3:Er, Yb nanophosphors (NPs) as imaging probes that emit luminescence excited by both near-infrared (NIR) light and an electron beam. Under 980 nm NIR light irradiation, the Y2O3:Tm, Yb and Y2O3:Er, Yb NPs emitted NIR luminescence (NIRL) around 810 nm and 1530 nm, respectively, and cathodoluminescence at 455 nm and 660 nm under excitation of accelerated electrons, respectively. Multimodalities of the NPs were confirmed in correlative NIRL/CL imaging and their locations were visualized at the same observation area in both NIRL and CL images. Using CL microscopy, the NPs were visualized at the single-particle level and with multicolour. Multiscale NIRL/CL bioimaging was demonstrated through in vivo and in vitro NIRL deep-tissue observations, cellular NIRL imaging, and high-spatial resolution CL imaging of the NPs inside cells. The location of a cell sheet transplanted onto the back muscle fascia of a hairy rat was visualized through NIRL imaging of the Y2O3:Er, Yb NPs. Accurate positions of cells through the thickness (1.5 mm) of a tissue phantom were detected by NIRL from the Y2O3:Tm, Yb NPs. Further, locations of the two types of NPs inside cells were observed using CL microscopy. PMID:27185264

Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 1; A Study Employing Neural Cell Adhesion Molecules (N-CAM) Immunocytochemistry and Conventional Morphological Techniques (Light and Electron Microscopy)

NASA Technical Reports Server (NTRS)

Daunton, N. G.; DAmelio, F.; Wu, L.; Ilyina-Kakueva, E. I.; Krasnov, I. B.; Hyde, T. M.; Sigworth, S. K.

1994-01-01

The effects of spaceflight upon the 'slow' muscle adductor longus was examined in rats flown in the Soviet Biosatellite COSMOS 2044. Three groups - synchronous, vivarium and basal served as controls. The techniques employed included standard methods for light microscopy, N-CAM immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy, contraction bands and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leucocytes and mononuclear cells. N-CAM immunoreactivity was seen (N-CAM-IR) on the myofiber surface, satellite cells and in regenerating myofibers reminiscent of myotubes. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments that displayed varied distributive patterns. The principal electron microscopic changes of the neuromuscular junctions consisted of a decrease or absence of synaptic vesicles, degeneration of axon terminals, increased number of microtubules, vacant axonal spaces and axonal sprouting. The present observations indicate that major alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations.

PubMed

Taylor, Michael A; Vanwalleghem, Gilles C; Favre-Bulle, Itia A; Scott, Ethan K

2018-06-19

Light-sheet microscopy is used extensively in developmental biology and neuroscience. One limitation of this approach is that absorption and scattering produces shadows in the illuminating light sheet, resulting in stripe artifacts. Here, we introduce diffuse light-sheet microscopes that use a line diffuser to randomize the light propagation within the image plane, allowing the light sheets to reform after obstacles. We incorporate diffuse light sheets in two existing configurations: selective plane illumination microscopy (SPIM) in which the sample is illuminated with a static sheet of light, and digitally scanned light sheet (DSLS) in which a thin Gaussian beam is scanned across the image plane during each acquisition. We compare diffuse light-sheet microscopes to their conventional counterparts for calcium imaging of neural activity in larval zebrafish. We show that stripe artifacts can cast deep shadows that conceal some neurons, and that the stripes can flicker, producing spurious signals that could be interpreted as biological activity. Diffuse light sheets mitigate these problems, illuminating the blind spots produced by stripes and removing artifacts produced by the stripes' movements. The upgrade to diffuse light sheets is simple and inexpensive, especially in the case of DSLS, where it requires the addition of one optical element. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Neuronal connectome of a sensory-motor circuit for visual navigation

PubMed Central

Randel, Nadine; Asadulina, Albina; Bezares-Calderón, Luis A; Verasztó, Csaba; Williams, Elizabeth A; Conzelmann, Markus; Shahidi, Réza; Jékely, Gáspár

2014-01-01

Animals use spatial differences in environmental light levels for visual navigation; however, how light inputs are translated into coordinated motor outputs remains poorly understood. Here we reconstruct the neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis using serial-section transmission electron microscopy. In this 71-neuron circuit, photoreceptors connect via three layers of interneurons to motorneurons, which innervate trunk muscles. By combining eye ablations with behavioral experiments, we show that the circuit compares light on either side of the body and stimulates body bending upon left-right light imbalance during visual phototaxis. We also identified an interneuron motif that enhances sensitivity to different light intensity contrasts. The Platynereis eye circuit has the hallmarks of a visual system, including spatial light detection and contrast modulation, illustrating how image-forming eyes may have evolved via intermediate stages contrasting only a light and a dark field during a simple visual task. DOI: http://dx.doi.org/10.7554/eLife.02730.001 PMID:24867217

Shedding new light on lipid functions with CARS and SRS microscopy

PubMed Central

Yu, Yong; Ramachandran, Prasanna V.; Wang, Meng C.

2014-01-01

Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. PMID:24576891

Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

PubMed

Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian

2016-10-01

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Effects of radiation upon the light-sensing elements of the retina as characterized by scanning electron microscopy

NASA Technical Reports Server (NTRS)

Malachowski, M. J.; Tobias, C. A.; Leith, J. T.

1977-01-01

A model system using Necturus maculosus, the common mudpuppy, was established for evaluating effects of radiation upon the light-sensing elements of the retina. Accelerated heavy ions of helium and neon from the Berkeley Bevalac were used. A number of criteria were chosen to characterize radiation damage by observing morphological changes with the scanning electron microscope. The studies indicated retina sensitivity to high-LET (neon) particles at radiation levels below 10 rads (7 particles per visual element) whereas no significant effects were seen from fast helium ions below 50 rads.

Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage.

PubMed

Castejon, O J; Castejon, H V; Diaz, M; Castellano, A

2001-10-01

Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.

Microstructure of milk

USDA-ARS?s Scientific Manuscript database

The fat and protein in milk may be examined by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy, and any bacteria present may be viewed by light microscopy. The fat exists as globules, the bulk of the protein is in the form of casein micelles, a...

Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme

PubMed Central

Woringer, Maxime; Darzacq, Xavier; Zimmer, Christophe

2017-01-01

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy. PMID:28788909

Camera array based light field microscopy

PubMed Central

Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

2015-01-01

This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

PubMed Central

Fu, Qinyi; Martin, Benjamin L.; Matus, David Q.; Gao, Liang

2016-01-01

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

Superresolution microscopy for microbiology

PubMed Central

Coltharp, Carla; Xiao, Jie

2014-01-01

Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments. PMID:22947061

The Light Microscopy Module: An On-Orbit Multi-User Microscope Facility

NASA Technical Reports Server (NTRS)

Motil, Susan M.; Snead, John H.

2002-01-01

The Light Microscopy Module (LMM) is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and operation of fluids and biology experiments within the Fluids and Combustion Facility (FCF) Fluids Integrated Rack (FIR) on the International Space Station (ISS). The LMM will be the first integrated payload with the FIR to conduct four fluid physics experiments. A description of the LMM diagnostic capabilities, including video microscopy, interferometry, laser tweezers, confocal, and spectrophotometry, will be provided.

Accessible microscopy workstation for students and scientists with mobility impairments.

PubMed

Duerstock, Bradley S

2006-01-01

An integrated accessible microscopy workstation was designed and developed to allow persons with mobility impairments to control all aspects of light microscopy with minimal human assistance. This system, named AccessScope, is capable of performing brightfield and fluorescence microscopy, image analysis, and tissue morphometry requisite for undergraduate science courses to graduate-level research. An accessible microscope is necessary for students and scientists with mobility impairments to be able to use a microscope independently to better understand microscopical imaging concepts and cell biology. This knowledge is not always apparent by simply viewing a catalog of histological images. The ability to operate a microscope independently eliminates the need to hire an assistant or rely on a classmate and permits one to take practical laboratory examinations by oneself. Independent microscope handling is also crucial for graduate students and scientists with disabilities to perform scientific research. By making a personal computer as the user interface for controlling AccessScope functions, different upper limb mobility impairments could be accommodated by using various computer input devices and assistive technology software. Participants with a range of upper limb mobility impairments evaluated the prototype microscopy workstation. They were able to control all microscopy functions including loading different slides without assistance.

Application of SEM and EDX in studying biomineralization in plant tissues.

PubMed

He, Honghua; Kirilak, Yaowanuj

2014-01-01

This chapter describes protocols using formalin-acetic acid-alcohol (FAA) to fix plant tissues for studying biomineralization by means of scanning electron microscopy (SEM) and qualitative energy-dispersive X-ray microanalysis (EDX). Specimen preparation protocols for SEM and EDX mainly include fixation, dehydration, critical point drying (CPD), mounting, and coating. Gold-coated specimens are used for SEM imaging, while gold- and carbon-coated specimens are prepared for qualitative X-ray microanalyses separately to obtain complementary information on the elemental compositions of biominerals. During the specimen preparation procedure for SEM, some biominerals may be dislodged or scattered, making it difficult to determine their accurate locations, and light microscopy is used to complement SEM studies. Specimen preparation protocols for light microscopy generally include fixation, dehydration, infiltration and embedding with resin, microtome sectioning, and staining. In addition, microwave processing methods are adopted here to speed up the specimen preparation process for both SEM and light microscopy.

Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bruns, Thomas; Schickinger, Sarah; Wittig, Rainer; Schneckenburger, Herbert

2012-10-01

A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy.

PubMed

Gualda, Emilio J; Simão, Daniel; Pinto, Catarina; Alves, Paula M; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

Impact of New Camera Technologies on Discoveries in Cell Biology.

PubMed

Stuurman, Nico; Vale, Ronald D

2016-08-01

New technologies can make previously invisible phenomena visible. Nowhere is this more obvious than in the field of light microscopy. Beginning with the observation of "animalcules" by Antonie van Leeuwenhoek, when he figured out how to achieve high magnification by shaping lenses, microscopy has advanced to this day by a continued march of discoveries driven by technical innovations. Recent advances in single-molecule-based technologies have achieved unprecedented resolution, and were the basis of the Nobel prize in Chemistry in 2014. In this article, we focus on developments in camera technologies and associated image processing that have been a major driver of technical innovations in light microscopy. We describe five types of developments in camera technology: video-based analog contrast enhancement, charge-coupled devices (CCDs), intensified sensors, electron multiplying gain, and scientific complementary metal-oxide-semiconductor cameras, which, together, have had major impacts in light microscopy. © 2016 Marine Biological Laboratory.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

PubMed Central

Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

Effect of Annealing Time of YAG:Ce3+ Phosphor on White Light Chromaticity Values

NASA Astrophysics Data System (ADS)

Abd, Husnen R.; Hassan, Z.; Ahmed, Naser M.; Almessiere, Munirah Abdullah; Omar, A. F.; Alsultany, Forat H.; Sabah, Fayroz A.; Osman, Ummu Shuhada

2018-02-01

Yttrium and aluminium nitrate phosphors doped with cerium nitrate and mixed with urea (fuel) are prepared by using microwave-induced combustion synthesis according to the formula Y(3-0.06)Al5O12:0.06Ce3+ (YAG:Ce3+) to produce white light emitting diodes by conversion from blue indium gallium nitride-light emitting diode chips. The sintering time with fixed temperature (1050°C) for phosphor powder was optimized and found to be 5 h. The crystallinity, structure, chemical composition, luminescent properties with varying currents densities and chromaticity were characterized by x-ray diffraction, field emission-scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, photoluminescence emission, electroluminescence and standard CIE 1931 chromaticity diagram, respectively. The energy levels of Ce3+ in YAG were discussed based on its absorption and excitation spectra. The results show that the obtained YAG:Ce3+ phosphor sintered for 5 h has good crystallinity with pure phase, low agglomerate with spherical shaped particles and strong yellow emission, offering cool-white LED with tuneable correlated color temperature and a good color rendering index compared to those prepared by sintering for 2 h and as-prepared phosphor powders.

Chemical analyses of fossil bone.

PubMed

Zheng, Wenxia; Schweitzer, Mary Higby

2012-01-01

The preservation of microstructures consistent with soft tissues, cells, and other biological components in demineralized fragments of dinosaur bone tens of millions of years old was unexpected, and counter to current hypotheses of tissue, cellular, and molecular degradation. Although the morphological similarity of these tissues to extant counterparts was unmistakable, after at least 80 million years exposed to geochemical influences, morphological similarity is insufficient to support an endogenous source. To test this hypothesis, and to characterize these materials at a molecular level, we applied multiple independent chemical, molecular, and microscopic analyses to identify the presence of original components produced by the extinct organisms. Microscopic techniques included field emission scanning electron microscopy, analytical transmission electron microscopy, transmitted light microscopy (LM), and fluorescence microscopy (FM). The chemical and molecular techniques include enzyme-linked immunosorbant assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot (immunoblot), and attenuated total reflectance infrared spectroscopy. In situ analyses performed directly on tissues included immunohistochemistry and time-of-flight secondary ion mass spectrometry. The details of sample preparation and methodology are described in detail herein.

Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

NASA Astrophysics Data System (ADS)

Liu, Yingshuai; Li, Xuelian; Bao, Shujuan; Lu, Zhisong; Li, Qing; Li, Chang Ming

2013-05-01

Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml-1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.

FIR Light Microscopy Module Set Up

NASA Image and Video Library

2009-11-09

ISS021-E-022457 (9 Nov. 2009) --- NASA astronaut Nicole Stott, Expedition 21 flight engineer, uses a communication system while installing the Light Microscopy Module (LMM) Spindle Bracket Assembly in the Fluids Integrated Rack (FIR) in the Destiny laboratory of the International Space Station. Canadian Space Agency astronaut Robert Thirsk (out of frame) assisted Stott.

Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas.

PubMed

Dhaliwal, Jasmeet S; Kaufman, Stephen C

2009-01-01

The purpose of this study was to evaluate morphological changes induced by corneal collagen cross-linking in a human ex vivo cornea, using confocal, electron, and light microscopy. The central epithelium was partially removed from ex vivo human corneal buttons. Riboflavin 0.1% solution was applied before ultraviolet A light treatment and then for every 2 minutes for 30 minutes while the corneas were exposed to ultraviolet A light at a wavelength of 370 nm and intensity of 3 mW/cm(2). Each cornea was evaluated using confocal, electron, and light microscopy. Confocal microscopy demonstrated normal-appearing corneas on their initial pretreatment examination, with reduced stromal detail. After treatment, a superficial layer of highly reflective spherical structures (4-10 microm) was observed. Many of these hyperreflective structures appeared up to a depth of 300 microm. The remainder of the corneal stroma and endothelium appeared normal. Electron microscopy showed keratocyte apoptotic changes to a depth of 300 microm. No observable pathologic changes were seen on histology. Based on clinical studies, corneal cross-linking is a promising treatment that appears to be safe and to halt ectatic corneal disease progression. Initial European studies used animal models to extrapolate human protocols. In conjunction with clinical studies, we believe that human ex vivo corneal studies provide a means to evaluate the structural and morphological changes associated with this procedure, within human corneas, in a manner that cannot be accomplished in vivo.

eduSPIM: Light Sheet Microscopy in the Museum.

PubMed

Jahr, Wiebke; Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.

X-ray micro-modulated luminescence tomography (XMLT)

PubMed Central

Cong, Wenxiang; Liu, Fenglin; Wang, Chao; Wang, Ge

2014-01-01

Imaging depth of optical microscopy has been fundamentally limited to millimeter or sub-millimeter due to strong scattering of light in a biological sample. X-ray microscopy can resolve spatial details of few microns deep inside a sample but contrast resolution is inadequate to depict heterogeneous features at cellular or sub-cellular levels. To enhance and enrich biological contrast at large imaging depth, various nanoparticles are introduced and become essential to basic research and molecular medicine. Nanoparticles can be functionalized as imaging probes, similar to fluorescent and bioluminescent proteins. LiGa5O8:Cr3+ nanoparticles were recently synthesized to facilitate luminescence energy storage with x-ray pre-excitation and subsequently stimulated luminescence emission by visible/near-infrared (NIR) light. In this paper, we propose an x-ray micro-modulated luminescence tomography (XMLT, or MLT to be more general) approach to quantify a nanophosphor distribution in a thick biological sample with high resolution. Our numerical simulation studies demonstrate the feasibility of the proposed approach. PMID:24663898

Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells

PubMed Central

2012-01-01

Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519–17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types. PMID:22967319

Holographic microscopy for in situ studies of microorganism motility

NASA Astrophysics Data System (ADS)

Nadeau, J.; Hu, S.; Jericho, S.; Lindensmith, C.

2011-12-01

Robust technologies for the detection and identification of microorganisms at low concentrations in complex liquid media are needed for numerous applications: environmental and medical microbiology, food safety, and for the search for microbial life elsewhere in the Solar System. The best current method for microbial enumeration is specific labeling with fluorescent dyes followed by high-resolution light microscopy. However, fluorescent techniques are difficult to use in situ in extreme environments (such as the Arctic and Antarctic or the open ocean) due to the fragility of the instruments and their high power demands. In addition, light microscopic techniques rarely provide insight into microbial motility behaviors. Tracking single cells would provide important insight into the physics of micron-scale motility as well as into key microbial phenomena such as surface attachment and invasiveness. An alternative to traditional light microscopy that is attracting increasing attention is holographic microscopy. Holographic microscopy works by illuminating the object of interest with coherent light from a laser. The light reflected from (or transmitted through) the object is then combined with a coherent reference beam to create an interference pattern that contains the phase and intensity information required to reconstruct a three dimensional image of the object. The interference pattern is recorded on a high resolution detector and can be used to computationally reconstruct a 3D image of the object. The lateral resolution of the image depends upon the wavelength of the light used, the laser power, camera quality, and external noise sources (vibration, stray light, and so forth). Although the principle is simple, technological barriers have prevented wider use of holographic microscopy. Laser sources and CCD cameras with the appropriate properties have only very recently become affordable. In addition, holographic microscopy leads to large data sets that are computationally intensive to reconstruct images from, so the technology to store and process large amounts of data are required. We have successfully deployed a digital in-line holographic microscope in lakes of the Canadian High Arctic and the open ocean. We present characteristic data sets from these experiments, as well as discussing how data acquisition and instrumentation can be improved. A design for a new type of autonomous, submersible holographic microscope incorporating an off-axis reference beam is presented, and future plans for controlled microbe-polymer studies are detailed.

Performance comparison of CareStart™ HRP2/pLDH combo rapid malaria test with light microscopy in north-western Tigray, Ethiopia: a cross-sectional study.

PubMed

Feleke, Daniel Getacher; Tarko, Shambel; Hadush, Haftom

2017-06-06

Rapid diagnostic tests (RDTs) are alternative methods for microscopy in the diagnosis of malaria in resource limited settings. Among commercially available RDTs, CareStart™ Malaria test was found to show reliable results. This study evaluated the performance of CareStart™ Malaria Combo test kit in Northwestern Tigray in Ethiopia. Blood samples were collected from 320 malaria-suspected patients at Mayani Hospital in Northwestern Tigray from December 2015 to March 2016. All blood samples were examined using both light microscopy and CareStart™ Malaria HRP2/pLDH Combo Test kit. Statistical analyses were performed using SPSS version 20. The overall parasite positivity using light microscopy and CareStart™ RDT was 41 (12.8%) and 43 (13.4%), respectively. The sensitivity and specificity of CareStart™ RDT, regardless of species, were found to be 95.4 and 99.3%, respectively. Furthermore, the sensitivity of CareStart™ RDT for Plasmodium falciparum or mixed infection and non-falciparum malaria parasites was 94.4 and 85.0%, respectively while the specificity was found to be 98.9 and 99.7%, respectively. The agreement between the two test methods was "excellent" with a kappa value of 0.92. CareStart™ RDT has very good sensitivity and specificity for malaria diagnosis. The test kit also has an excellent agreement with light microscopy. It is therefore useful in resource-limited areas where microscopy is not available.

Accessible Microscopy Workstation for Students and Scientists with Mobility Impairments

ERIC Educational Resources Information Center

Duerstock, Bradley S.

2006-01-01

An integrated accessible microscopy workstation was designed and developed to allow persons with mobility impairments to control all aspects of light microscopy with minimal human assistance. This system, named AccessScope, is capable of performing brightfield and fluorescence microscopy, image analysis, and tissue morphometry requisite for…

Noise reduction in digital lensless holographic microscopy by engineering the light from a light-emitting diode.

PubMed

Garcia-Sucerquia, Jorge

2013-01-01

By engineering the light from a light-emitting diode (LED) the noises present in digital lensless holographic microscopy (DLHM) are reduced. The partially coherent light from an LED is tailored to produce a spherical wavefront with limited coherence time and the spatial coherence needed by DLHM to work. DLHM with this engineered light source is used to image biological samples that cover areas of the order of mm(2). The ratio between the diameter of the area that is almost coherently illuminated to the diameter of the illumination area is utilized as parameter to quantify the performance of the DLHM with the engineered LED light source. Experimental results show that while the noises can be reduced effectively the spatial resolution can be kept in the micrometer range.

A novel fibrous duct structure discovered in the brain meninges by using polarized light microscopy

NASA Astrophysics Data System (ADS)

Nam, Min-Ho; Jung, Sharon Jiyoon; Soh, Kwang-Sup; Lim, Jaekwan; Seo, Eunseok; Lim, Jun; Baek, Miok; Lee, Sang Joon

2016-05-01

We have previously reported the discovery of a novel fibrous structure (NFS) consisting of unidirectionally arranged collagen fibers in the spinal pia mater. Due to its unique structure, it was easily detected using polarized light microscopy. In the current study, we describe the discovery of a similar NFS in the brain meninges of rats by using polarized light microscopy. This NFS is located beneath the superior sagittal sinus. Initially, we systemically analyzed the polarization properties of the NFS. The change in the light intensity of the NFS, with respect to the polarization angle, was eight times greater than that of blood vessels, showing that the collagen fibers are oriented in a particular direction with almost perfect parallelism (0.99). The orientation angle of the polarization ellipse confirmed the orientation of the collagen fibers in the NFS. Histological studies further confirmed that the unidirectionally arranged collagen fibers were responsible for this distinct polarization property. Surprisingly, X-ray microtomography and 3D confocal imaging revealed that the NFS contains within it a duct structure, a putative primo vessel. In conclusion, we report a NFS in the brain meninges, detected by using polarized light microscopy, that provides space for a putative primo vessel, not a blood vessel.

The evolution of structured illumination microscopy in studies of HIV.

PubMed

Marno, Kelly; Al'Zoubi, Lara; Pearson, Matthew; Posch, Markus; McKnight, Áine; Wheeler, Ann P

2015-10-15

The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years. Copyright © 2015. Published by Elsevier Inc.

Laser Light-field Fusion for Wide-field Lensfree On-chip Phase Contrast Microscopy of Nanoparticles

NASA Astrophysics Data System (ADS)

Kazemzadeh, Farnoud; Wong, Alexander

2016-12-01

Wide-field lensfree on-chip microscopy, which leverages holography principles to capture interferometric light-field encodings without lenses, is an emerging imaging modality with widespread interest given the large field-of-view compared to lens-based techniques. In this study, we introduce the idea of laser light-field fusion for lensfree on-chip phase contrast microscopy for detecting nanoparticles, where interferometric laser light-field encodings acquired using a lensfree, on-chip setup with laser pulsations at different wavelengths are fused to produce marker-free phase contrast images of particles at the nanometer scale. As a proof of concept, we demonstrate, for the first time, a wide-field lensfree on-chip instrument successfully detecting 300 nm particles across a large field-of-view of ~30 mm2 without any specialized or intricate sample preparation, or the use of synthetic aperture- or shift-based techniques.

Laser Light-field Fusion for Wide-field Lensfree On-chip Phase Contrast Microscopy of Nanoparticles.

PubMed

Kazemzadeh, Farnoud; Wong, Alexander

2016-12-13

Wide-field lensfree on-chip microscopy, which leverages holography principles to capture interferometric light-field encodings without lenses, is an emerging imaging modality with widespread interest given the large field-of-view compared to lens-based techniques. In this study, we introduce the idea of laser light-field fusion for lensfree on-chip phase contrast microscopy for detecting nanoparticles, where interferometric laser light-field encodings acquired using a lensfree, on-chip setup with laser pulsations at different wavelengths are fused to produce marker-free phase contrast images of particles at the nanometer scale. As a proof of concept, we demonstrate, for the first time, a wide-field lensfree on-chip instrument successfully detecting 300 nm particles across a large field-of-view of ~30 mm 2 without any specialized or intricate sample preparation, or the use of synthetic aperture- or shift-based techniques.

New approach for the quantification of processed animal proteins in feed using light microscopy.

PubMed

Veys, P; Baeten, V

2010-07-01

A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.

Methods and apparatus of spatially resolved electroluminescence of operating organic light-emitting diodes using conductive atomic force microscopy

NASA Technical Reports Server (NTRS)

Hersam, Mark C. (Inventor); Pingree, Liam S. C. (Inventor)

2008-01-01

A conductive atomic force microscopy (cAFM) technique which can concurrently monitor topography, charge transport, and electroluminescence with nanometer spatial resolution. This cAFM approach is particularly well suited for probing the electroluminescent response characteristics of operating organic light-emitting diodes (OLEDs) over short length scales.

Light Microscopy of the Hair: A Simple Tool to “Untangle” Hair Disorders

PubMed Central

Adya, Keshavmurthy A; Inamadar, Arun C; Palit, Aparna; Shivanna, Ragunatha; Deshmukh, Niranjan S

2011-01-01

Light microscopy of the hair forms an important bedside clinical tool for the diagnosis of various disorders affecting the hair. Hair abnormalities can be seen in the primary diseases affecting the hair or as a secondary involvement of hair in diseases affecting the scalp. Hair abnormalities also form a part of various genodermatoses and syndromes. In this review, we have briefly highlighted the light microscopic appearance of various infectious and non-infectious conditions affecting the hair. PMID:21769242

Damage threshold in adult rabbit eyes after scleral cross-linking by riboflavin/blue light application.

PubMed

Iseli, Hans Peter; Körber, Nicole; Karl, Anett; Koch, Christian; Schuldt, Carsten; Penk, Anja; Liu, Qing; Huster, Daniel; Käs, Josef; Reichenbach, Andreas; Wiedemann, Peter; Francke, Mike

2015-10-01

Several scleral cross-linking (SXL) methods were suggested to increase the biomechanical stiffness of scleral tissue and therefore, to inhibit axial eye elongation in progressive myopia. In addition to scleral cross-linking and biomechanical effects caused by riboflavin and light irradiation such a treatment might induce tissue damage, dependent on the light intensity used. Therefore, we characterized the damage threshold and mechanical stiffening effect in rabbit eyes after application of riboflavin combined with various blue light intensities. Adult pigmented and albino rabbits were treated with riboflavin (0.5 %) and varying blue light (450 ± 50 nm) dosages from 18 to 780 J/cm(2) (15 to 650 mW/cm(2) for 20 min). Scleral, choroidal and retinal tissue alterations were detected by means of light microscopy, electron microscopy and immunohistochemistry. Biomechanical changes were measured by shear rheology. Blue light dosages of 480 J/cm(2) (400 mW/cm(2)) and beyond induced pathological changes in ocular tissues; the damage threshold was defined by the light intensities which induced cellular degeneration and/or massive collagen structure changes. At such high dosages, we observed alterations of the collagen structure in scleral tissue, as well as pigment aggregation, internal hemorrhages, and collapsed blood vessels. Additionally, photoreceptor degenerations associated with microglia activation and macroglia cell reactivity in the retina were detected. These pathological alterations were locally restricted to the treated areas. Pigmentation of rabbit eyes did not change the damage threshold after a treatment with riboflavin and blue light but seems to influence the vulnerability for blue light irradiations. Increased biomechanical stiffness of scleral tissue could be achieved with blue light intensities below the characterized damage threshold. We conclude that riboflavin and blue light application increased the biomechanical stiffness of scleral tissue at blue light energy levels below the damage threshold. Therefore, applied blue light intensities below the characterized damage threshold might define a therapeutic blue light tolerance range. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Polymer Blends: Optical Microscopy (*Polarized, Interference and Phase Contrast Microscopy*) and Confocal Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramanathan, Nathan Muruganathan; Darling, Seth B.

2015-01-01

Chapter 15 surveys the characterization of macro, micro and meso morphologies of polymer blends by optical microscopy. Confocal Microscopy offers the ability to view the three dimensional morphology of polymer blends, popular in characterization of biological systems. Confocal microscopy uses point illumination and a spatial pinhole to eliminate out-of focus light in samples that are thicker than the focal plane.

Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

2017-02-01

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Microscopic analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) embryonic development before and after treatment with azadirachtin, lufenuron, and deltamethrin.

PubMed

Correia, Alicely A; Wanderley-Teixeira, Valéria; Teixeira, Alvaro A C; Oliveira, José V; Gonçalves, Gabriel G A; Cavalcanti, MaríIia G S; Brayner, Fábio A; Alves, Luiz C

2013-04-01

The botanical insecticides, growth regulators, and pyrethroids have an effect on the biology of Spodoptera frugiperda (Smith). However, no emphasis has been given to the effect of these insecticides on embryonic development of insects, in histological level. Thus, this research aimed to examine by light and scanning electron microscopy S. frugiperda eggs and to describe the embryonic development, before and after immersion treatment, using commercial concentrations and lower concentrations than commercial ones, of the compounds lufenuron (Match), azadirachtin (AzaMax), and deltamethrin (Decis-positive control). For light microscopy semithin sections of eggs were used, and for scanning electron microscopy, images of the surface of eggs, treated and untreated with insecticides. The morphological characteristics of S. frugiperda eggs, in general, were similar to those described in the literature for most of the insects in the order Lepidoptera. Spherical eggs slightly flattened at the poles, with chorion, yolk, vitelline membrane, and embryo formation. In both microscopic analysis, we observed that insecticides acted immediately and independent of concentration, resulting absence, or incomplete embryo, presented yolk granules widely dispersed, without vitellophage formation, chorion disintegration, disorganized blastoderm, presenting vacuoles, yolk region with amorphous cells, and formation of completely uncharacterized appendages. Thus, we conclude that the compounds lufenuron and azadirachtin interfere on S. frugiperda embryonic development.

Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode.

PubMed

Poher, V; Zhang, H X; Kennedy, G T; Griffin, C; Oddos, S; Gu, E; Elson, D S; Girkin, M; French, P M W; Dawson, M D; Neil, M A

2007-09-03

We describe an optical sectioning microscopy system with no moving parts based on a micro-structured stripe-array light emitting diode (LED). By projecting arbitrary line or grid patterns onto the object, we are able to implement a variety of optical sectioning microscopy techniques such as grid-projection structured illumination and line scanning confocal microscopy, switching from one imaging technique to another without modifying the microscope setup. The micro-structured LED and driver are detailed and depth discrimination capabilities are measured and calculated.

Imaging three-dimensional light propagation through periodic nanohole arrays using scanning aperture microscopy

PubMed Central

Chowdhury, Mustafa H.; Catchmark, Jeffrey M.; Lakowicz, Joseph R.

2009-01-01

The authors introduce a technique for three-dimensional (3D) imaging of the light transmitted through periodic nanoapertures using a scanning probe to perform optical sectioning microscopy. For a 4×4 nanohole array, the transmitted light displays intensity modulations along the propagation axis, with the maximum intensity occurring at 450 μm above the surface. The propagating fields show low divergence, suggesting a beaming effect induced by the array. At distances within 25 μm from the surface, they observe subwavelength confinement of light propagating from the individual nanoholes. Hence, this technique can potentially be used to map the 3D distribution of propagating light, with high spatial resolution. PMID:19696912

Rapid diagnosis of tinea incognito using handheld reflectance confocal microscopy: a paradigm shift in dermatology?

PubMed

Navarrete-Dechent, Cristián; Bajaj, Shirin; Marghoob, Ashfaq A; Marchetti, Michael A

2015-06-01

Dermatophytoses are common skin infections. Traditional diagnostic tests such as skin scrapings for light microscopy examination, fungal cultures and biopsies remain imperfect due to false-negative test results, cost, time required to perform the procedure, time delays in test results and/or a requirement for an invasive procedure. Herein, we present a case of an 80-year-old female whose tinea incognito was non-invasively diagnosed within seconds using handheld reflectance confocal microscopy (RCM). As non-invasive skin imaging continues to improve, we expect light-based office microscopy to be replaced with technologies such as RCM, which has multiple and continually expanding diagnostic applications. © 2015 Blackwell Verlag GmbH.

Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms (Phoenicococcus marlatti Ckll., 1899).

PubMed

Asensio, L; Lopez-Llorca, L V; López-Jiménez, J A

2005-01-01

We have evaluated the parasitism of the red scale insect of the date palm (Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves (Phoenix dactylifera L.) and did not parasite the scale insects.

Standardization for Ki-67 Assessment in Moderately Differentiated Breast Cancer. A Retrospective Analysis of the SAKK 28/12 Study

PubMed Central

Varga, Zsuzsanna; Cassoly, Estelle; Li, Qiyu; Oehlschlegel, Christian; Tapia, Coya; Lehr, Hans Anton; Klingbiel, Dirk; Thürlimann, Beat; Ruhstaller, Thomas

2015-01-01

Background Proliferative activity (Ki-67 Labelling Index) in breast cancer increasingly serves as an additional tool in the decision for or against adjuvant chemotherapy in midrange hormone receptor positive breast cancer. Ki-67 Index has been previously shown to suffer from high inter-observer variability especially in midrange (G2) breast carcinomas. In this study we conducted a systematic approach using different Ki-67 assessments on large tissue sections in order to identify the method with the highest reliability and the lowest variability. Materials and Methods Five breast pathologists retrospectively analyzed proliferative activity of 50 G2 invasive breast carcinomas using large tissue sections by assessing Ki-67 immunohistochemistry. Ki-67-assessments were done on light microscopy and on digital images following these methods: 1) assessing five regions, 2) assessing only darkly stained nuclei and 3) considering only condensed proliferative areas (‘hotspots’). An individual review (the first described assessment from 2008) was also performed. The assessments on light microscopy were done by estimating. All measurements were performed three times. Inter-observer and intra-observer reliabilities were calculated using the approach proposed by Eliasziw et al. Clinical cutoffs (14% and 20%) were tested using Fleiss’ Kappa. Results There was a good intra-observer reliability in 5 of 7 methods (ICC: 0.76–0.89). The two highest inter-observer reliability was fair to moderate (ICC: 0.71 and 0.74) in 2 methods (region-analysis and individual-review) on light microscopy. Fleiss’-kappa-values (14% cut-off) were the highest (moderate) using the original recommendation on light-microscope (Kappa 0.58). Fleiss’ kappa values (20% cut-off) were the highest (Kappa 0.48 each) in analyzing hotspots on light-microscopy and digital-analysis. No methodologies using digital-analysis were superior to the methods on light microscope. Conclusion Our results show that all methods on light-microscopy for Ki-67 assessment in large tissue sections resulted in a good intra-observer reliability. Region analysis and individual review (the original recommendation) on light-microscopy yielded the highest inter-observer reliability. These results show slight improvement to previously published data on poor-reproducibility and thus might be a practical-pragmatic way for routine assessment of Ki-67 Index in G2 breast carcinomas. PMID:25885288

Cassette Series Designed for Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast

PubMed Central

Young, Carissa L.; Raden, David L.; Caplan, Jeffrey; Czymmek, Kirk; Robinson, Anne S.

2012-01-01

During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization, and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize, and monitor the spatial localization of proteins and complexes at the sub-organelle level; yet, many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for FlAsH-based localization, and the first evaluation in yeast of a photoswitchable variant – mEos2 – to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolor labeling in vivo. Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif that is compatible with diaminobenzidine photooxidation used for protein localization by electron microscopy and mEos2 that is ideal for super-resolution microscopy. We have examined the utility of our cassettes by analyzing all probes fused to the C-terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live-cell, super-resolution or electron microscopy imaging techniques. PMID:22473760

Tyloses and Phenolic Deposits in Xylem Vessels Impede Water Transport in Low-Lignin Transgenic Poplars: A Study by Cryo-Fluorescence Microscopy1[W][OA

PubMed Central

Kitin, Peter; Voelker, Steven L.; Meinzer, Frederick C.; Beeckman, Hans; Strauss, Steven H.; Lachenbruch, Barbara

2010-01-01

Of 14 transgenic poplar genotypes (Populus tremula × Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water- or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse. PMID:20639405

The Building Blocks of Materials: Gathering Knowledge at the Molecular Level

NASA Technical Reports Server (NTRS)

2003-01-01

Two start-up positions were created within SD46 to pursue developments in the rapidly expanding areas of biomineralization and nano-technology. As envisioned by Dr. Sandor Lehoczy, the new laboratories to be developed must have the capacity to investigate not only processes associated with the self-assembly of molecules but also the examination of self-assembled structures. For these purposes, laboratories capable of performing the intended function, particularly light scattering spectroscopy and atomic force microscopy were created. What follows then are recent advances arising from the development of these new laboratories. With the implementation of the Atomic Force Microscopy Facility, examples of investigations that determine a correlation between the molecular structure of materials and their macroscopic physical properties are provided. In addition, examples of investigations with particular emphasis on the physical properties of protein crystals, at the molecular level, and subsequent macroscopic characteristics are as provided. Finally, progress in fabrication of technology at the nano-scale levels at the developmental stage is also presented.

Acquired Fanconi syndrome with proximal tubular cytoplasmic fibrillary inclusions of λ light chain restriction.

PubMed

Yao, Ying; Wang, Su-Xia; Zhang, You-Kang; Wang, Yan; Liu, Li; Liu, Gang

2014-01-01

Light chain proximal tubulopathy is a rarely reported entity associated with plasma cell dyscrasia that classically manifests as acquired Fanconi syndrome and is characterized by the presence of κ-restricted crystals in the proximal tubular cytoplasm. We herein present a case of multiple myeloma with Fanconi syndrome and acute kidney injury due to light chain proximal tubulopathy with light chain cast nephropathy. Prominent phagolysosomes and numerous irregularly shaped inclusions with a fibrillary matrix in the cytoplasm of the proximal tubules were identified on electron microscopy. A monotypic light chain of the λ type was detected in the distal tubular casts, proximal tubular cytoplasmic lysosomes and fibrillary inclusions on immunofluorescence and immune electron microscopy. This case underscores the importance of conducting careful ultrastructural investigations and immunocytologic examinations of light chains for detecting and diagnosing light chain proximal tubulopathy.

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

PubMed Central

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

2016-01-01

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

[Current approaches to evaluating the anatomic and functional status of the cornea].

PubMed

Avetisov, S E; Borodina, N V; Kobzova, M V; Musaeva, G M

2010-01-01

The review provides data on current methods for evaluating the anatomic and functional status of the cornea (light refraction, light transmission, and biomechanical properties, in particular). It analyzes the main advantages and disadvantages of basic (biomicroscopy, endothelial microscopy, ophthalmometry, topography, and pachymetry) and special (confocal microscopy, optical coherence tomography, ultrasound biomicroscopy, aberrometry, bidirectional corneal applanation, and keratoesthesiometry) studies.

Label-free, multi-scale imaging of ex-vivo mouse brain using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Min, Eunjung; Kandel, Mikhail E.; Ko, Chemyong J.; Popescu, Gabriel; Jung, Woonggyu; Best-Popescu, Catherine

2016-12-01

Brain connectivity spans over broad spatial scales, from nanometers to centimeters. In order to understand the brain at multi-scale, the neural network in wide-field has been visualized in detail by taking advantage of light microscopy. However, the process of staining or addition of fluorescent tags is commonly required, and the image contrast is insufficient for delineation of cytoarchitecture. To overcome this barrier, we use spatial light interference microscopy to investigate brain structure with high-resolution, sub-nanometer pathlength sensitivity without the use of exogenous contrast agents. Combining wide-field imaging and a mosaic algorithm developed in-house, we show the detailed architecture of cells and myelin, within coronal olfactory bulb and cortical sections, and from sagittal sections of the hippocampus and cerebellum. Our technique is well suited to identify laminar characteristics of fiber tract orientation within white matter, e.g. the corpus callosum. To further improve the macro-scale contrast of anatomical structures, and to better differentiate axons and dendrites from cell bodies, we mapped the tissue in terms of its scattering property. Based on our results, we anticipate that spatial light interference microscopy can potentially provide multiscale and multicontrast perspectives of gross and microscopic brain anatomy.

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples.

PubMed

Migliori, Bianca; Datta, Malika S; Dupre, Christophe; Apak, Mehmet C; Asano, Shoh; Gao, Ruixuan; Boyden, Edward S; Hermanson, Ola; Yuste, Rafael; Tomer, Raju

2018-05-29

Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.

Imaging a seizure model in zebrafish with structured illumination light sheet microscopy

NASA Astrophysics Data System (ADS)

Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Baraban, Scott; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

2018-02-01

Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

Gastroesophageal junction of Anatolian shepherd dog; a study by topographic anatomy, scanning electron and light microscopy.

PubMed

Alsafy, M A M; El-Gendy, S A A

2012-03-01

The aim of this study was to cast a spotlight on the topography and to point out the clinical importance of the gastroesophageal junction (GEJ) in Anatolian Shepherd dogs. Nine Anatolian Shepherd dogs were used to study the morphology of the GEJ. The esophagus was appeared has a portion within the thoracic cavity while no portion of the esophagus presented within the abdominal cavity that documented the absence of the intra-abdominal portion in all studied dogs. The topographic anatomy, scanning electron and light microscopic examinations revealed that the gastroesophageal junction was located at the level of the phrenico-esophageal ligament (PEL) inside the esophageal hiatus. Our results were distinguished the morphology of the esophageal and gastric cardiac mucosa at the level of the gastroesophageal junction by the scanning electron micrographs. The light microscopical examination was explained the PEL attached to the esophageal side in one dog and to the gastric cardiac side in three dogs.

Structure of the Global Nanoscience and Nanotechnology Research Literature

DTIC Science & Technology

2006-01-01

Transistors, Nature, 424 (6949): 654-657, 2003. Joannopoulos, JD, Meade, RD, Winn, JN, Photonic Crystals: Molding the Flow of Light, Princeton...1.27 Force Microscopy 40 0.10 0.00 Electron Spectroscopy 40 0.10 0.00 Rutherford backscattering spectrometry 38 0.10 0.00 flow cytometry 36 0.09...Backscattering Spectroscopy/Spectrometry • Flow Cytometry • Spectrophotometry (UV-Visible) • Deep Level Transient Spectroscopy • Inductively

Femtosecond digital lensless holographic microscopy to image biological samples.

PubMed

Mendoza-Yero, Omel; Calabuig, Alejandro; Tajahuerce, Enrique; Lancis, Jesús; Andrés, Pedro; Garcia-Sucerquia, Jorge

2013-09-01

The use of femtosecond laser radiation in digital lensless holographic microscopy (DLHM) to image biological samples is presented. A mode-locked Ti:Sa laser that emits ultrashort pulses of 12 fs intensity FWHM, with 800 nm mean wavelength, at 75 MHz repetition rate is used as a light source. For comparison purposes, the light from a light-emitting diode is also used. A section of the head of a drosophila melanogaster fly is studied with both light sources. The experimental results show very different effects of the pinhole size on the spatial resolution with DLHM. Unaware phenomena on the field of the DLHM are analyzed.

Quantitative tracking of tumor cells in phase-contrast microscopy exploiting halo artifact pattern

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

2012-03-01

Tumor cell morphology is closely related to its invasiveness characteristics and migratory behaviors. An invasive tumor cell has a highly irregular shape, whereas a spherical cell is non-metastatic. Thus, quantitative analysis of cell features is crucial to determine tumor malignancy or to test the efficacy of anticancer treatment. We use phase-contrast microscopy to analyze single cell morphology and to monitor its change because it enables observation of long-term activity of living cells without photobleaching and phototoxicity, which is common in other fluorescence-labeled microscopy. Despite this advantage, there are image-level drawbacks to phase-contrast microscopy, such as local light effect and contrast interference ring, among others. Thus, we first applied a local filter to compensate for non-uniform illumination. Then, we used intensity distribution information to detect the cell boundary. In phase-contrast microscopy images, the cell normally appears as a dark region surrounded by a bright halo. As the halo artifact around the cell body is minimal and has an asymmetric diffusion pattern, we calculated the cross-sectional plane that intersected the center of each cell and was orthogonal to the first principal axis. Then, we extracted the dark cell region by level set. However, a dense population of cultured cells still rendered single-cell analysis difficult. Finally, we measured roundness and size to classify tumor cells into malignant and benign groups. We validated segmentation accuracy by comparing our findings with manually obtained results.

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

PubMed

Shi, Hong-Bo; Sun, Hai-Qing; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Chen, De-Xi; Lou, Jin-Li; Duan, Zhong-Ping

2015-05-07

To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks

PubMed Central

2010-01-01

Background Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. Methods The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Results Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). Conclusions An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available. PMID:20459613

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks.

PubMed

Andrade, Bruno B; Reis-Filho, Antonio; Barros, Austeclino M; Souza-Neto, Sebastião M; Nogueira, Lucas L; Fukutani, Kiyoshi F; Camargo, Erney P; Camargo, Luís M A; Barral, Aldina; Duarte, Angelo; Barral-Netto, Manoel

2010-05-06

Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available.

White-light diffraction phase microscopy at doubled space-bandwidth product.

PubMed

Shan, Mingguang; Kandel, Mikhail E; Majeed, Hassaan; Nastasa, Viorel; Popescu, Gabriel

2016-12-12

White light diffraction microscopy (wDPM) is a quantitative phase imaging method that benefits from both temporal and spatial phase sensitivity, granted, respectively, by the common-path geometry and white light illumination. However, like all off-axis quantitative phase imaging methods, wDPM is characterized by a reduced space-bandwidth product compared to phase shifting approaches. This happens essentially because the ultimate resolution of the image is governed by the period of the interferogram and not just the diffraction limit. As a result, off-axis techniques generates single-shot, i.e., high time-bandwidth, phase measurements, at the expense of either spatial resolution or field of view. Here, we show that combining phase-shifting and off-axis, the original space-bandwidth is preserved. Specifically, we developed phase-shifting diffraction phase microscopy with white light, in which we measure and combine two phase shifted interferograms. Due to the white light illumination, the phase images are characterized by low spatial noise, i.e., <1nm pathlength. We illustrate the operation of the instrument with test samples, blood cells, and unlabeled prostate tissue biopsy.

Richardson-Lucy deconvolution as a general tool for combining images with complementary strengths.

PubMed

Ingaramo, Maria; York, Andrew G; Hoogendoorn, Eelco; Postma, Marten; Shroff, Hari; Patterson, George H

2014-03-17

We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timmermans, F. J.; Otto, C.

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemicallymore » or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.« less

Spectrally resolved laser interference microscopy

NASA Astrophysics Data System (ADS)

Butola, Ankit; Ahmad, Azeem; Dubey, Vishesh; Senthilkumaran, P.; Singh Mehta, Dalip

2018-07-01

We developed a new quantitative phase microscopy technique, namely, spectrally resolved laser interference microscopy (SR-LIM), with which it is possible to quantify multi-spectral phase information related to biological specimens without color crosstalk using a color CCD camera. It is a single shot technique where sequential switched on/off of red, green, and blue (RGB) wavelength light sources are not required. The method is implemented using a three-wavelength interference microscope and a customized compact grating based imaging spectrometer fitted at the output port. The results of the USAF resolution chart while employing three different light sources, namely, a halogen lamp, light emitting diodes, and lasers, are discussed and compared. The broadband light sources like the halogen lamp and light emitting diodes lead to stretching in the spectrally decomposed images, whereas it is not observed in the case of narrow-band light sources, i.e. lasers. The proposed technique is further successfully employed for single-shot quantitative phase imaging of human red blood cells at three wavelengths simultaneously without color crosstalk. Using the present technique, one can also use a monochrome camera, even though the experiments are performed using multi-color light sources. Finally, SR-LIM is not only limited to RGB wavelengths, it can be further extended to red, near infra-red, and infra-red wavelengths, which are suitable for various biological applications.

A national facility for biological cryo-electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saibil, Helen R., E-mail: h.saibil@mail.cryst.bbk.ac.uk; Grünewald, Kay; Stuart, David I.

2015-01-01

This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron. Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided ofmore » the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.« less

Gold-FISH: A correlative approach to microscopic imaging of single microbial cells in environmental samples

NASA Astrophysics Data System (ADS)

Schmidt, Hannes; Seki, David; Woebken, Dagmar; Eickhorst, Thilo

2017-04-01

Fluorescence in situ hybridization (FISH) is routinely used for the phylogenetic identification, detection, and quantification of single microbial cells environmental microbiology. Oligonucleotide probes that match the 16S rRNA sequence of target organisms are generally applied and the resulting signals are visualized via fluorescence microscopy. Consequently, the detection of the microbial cells of interest is limited by the resolution and the sensitivity of light microscopy where objects smaller than 0.2 µm can hardly be represented. Visualizing microbial cells at magnifications beyond light microscopy, however, can provide information on the composition and potential complexity of microbial habitats - the actual sites of nutrient cycling in soil and sediments. We present a recently developed technique that combines (1) the phylogenetic identification and detection of individual microorganisms by epifluorescence microscopy, with (2) the in situ localization of gold-labelled target cells on an ultrastructural level by SEM. Based on 16S rRNA targeted in situ hybridization combined with catalyzed reporter deposition, a streptavidin conjugate labeled with a fluorescent dye and nanogold particles is introduced into whole microbial cells. A two-step visualization process including an autometallographic enhancement of nanogold particles then allows for either fluorescence or electron microscopy, or a correlative application thereof. We will present applications of the Gold-FISH protocol to samples of marine sediments, agricultural soils, and plant roots. The detection and enumeration of bacterial cells in soil and sediment samples was comparable to CARD-FISH applications via fluorescence microscopy. Examples of microbe-surface interaction analysis will be presented on the basis of bacteria colonizing the rhizoplane of rice roots. In principle, Gold-FISH can be performed on any material to give a snapshot of microbe-surface interactions and provides a promising tool for the acquisition of correlative information on microorganisms within their respective habitats.

Asymmetrical flow field-flow fractionation with multi-angle light scattering and quasi-elastic light scattering for characterization of polymersomes: comparison with classical techniques.

PubMed

Till, Ugo; Gaucher-Delmas, Mireille; Saint-Aguet, Pascale; Hamon, Glenn; Marty, Jean-Daniel; Chassenieux, Christophe; Payré, Bruno; Goudounèche, Dominique; Mingotaud, Anne-Françoise; Violleau, Frédéric

2014-12-01

Polymersomes formed from amphiphilic block copolymers, such as poly(ethyleneoxide-b-ε-caprolactone) (PEO-b-PCL) or poly(ethyleneoxide-b-methylmethacrylate), were characterized by asymmetrical flow field-flow fractionation coupled with quasi-elastic light scattering (QELS), multi-angle light scattering (MALS), and refractive index detection, leading to the determination of their size, shape, and molecular weight. The method was cross-examined with more classical ones, like batch dynamic and static light scattering, electron microscopy, and atomic force microscopy. The results show good complementarities between all the techniques; asymmetrical flow field-flow fractionation being the most pertinent one when the sample exhibits several different types of population.

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

PubMed

Toda, Keisuke; Isobe, Keisuke; Namiki, Kana; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2018-04-01

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Pump-probe optical microscopy for imaging nonfluorescent chromophores.

PubMed

Wei, Lu; Min, Wei

2012-06-01

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light-molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.

Utility of fluorescence microscopy in embryonic/fetal topographical analysis.

PubMed

Zucker, R M; Elstein, K H; Shuey, D L; Ebron-McCoy, M; Rogers, J M

1995-06-01

For topographical analysis of developing embryos, investigators typically rely on scanning electron microscopy (SEM) to provide the surface detail not attainable with light microscopy. SEM is an expensive and time-consuming technique, however, and the preparation procedure may alter morphology and leave the specimen friable. We report that by using a high-resolution compound epifluorescence microscope with inexpensive low-power objectives and the fluorochrome acridine orange, we were able to obtain surface images of fixed or fresh whole rat embryos and fetal palates of considerably greater topographical detail than those obtained using routine light microscopy. Indeed the resulting high-resolution images afford not only superior qualitative documentation of morphological observations, but the capability for detailed morphometry via digitization and computer-assisted image analysis.

Quadriplegic areflexic ICU illness: selective thick filament loss and normal nerve histology.

PubMed

Sander, Howard W; Golden, Marianna; Danon, Moris J

2002-10-01

Areflexic quadriplegia that occurs in the intensive care unit (ICU) is commonly ascribed to critical illness polyneuropathy based upon electrophysiology or muscle light microscopy. However, electron microscopy often documents a selective thick filament loss myopathy. Eight ICU patients who developed areflexic quadriplegia underwent biopsy. Seven patients had received steroids, and 2 had also received paralytic agents. Electrodiagnostic studies revealed absent or low-amplitude motor responses in 7. Sensory responses were normal in 5 of 6 and absent in 1. Initial electromyography revealed absent (n = 3), small (n = 3), or polyphasic (n = 1) motor unit potentials, and diffuse fibrillation potentials (n = 5). In all 8, light microscopy of muscle revealed numerous atrophic-angulated fibers and corelike lesions, and electron microscopy revealed extensive thick filament loss. Morphology of sural and intramuscular nerves, and, in one autopsied case, of the obturator nerve and multiple nerve roots, was normal. Although clinical, electrodiagnostic, and light microscopic features mimicked denervating disease, muscle electron microscopy revealed thick filament loss, and nerve histology was normal. This suggests that areflexic ICU quadriplegia is a primary myopathy and not an axonal polyneuropathy. Copyright 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 499-505, 2002

The Fluids Integrated Rack and Light Microscopy Module Integrated Capabilities

NASA Technical Reports Server (NTRS)

Motil, Susan M.; Gati, Frank; Snead, John H.; Hill, Myron E.; Griffin, DeVon W.

2003-01-01

The Fluids Integrated Rack (FIR), a facility class payload, and the Light Microscopy Module (LMM), a subrack payload, are scheduled to be launched in 2005. The LMM integrated into the FIR will provide a unique platform for conducting fluids and biological experiments on ISS. The FIR is a modular, multi-user scientific research facility that will fly in the U.S. laboratory module, Destiny, of the International Space Station (ISS). The first payload in the FIR will be the Light Microscopy Module (LMM). The LMM is planned as a remotely controllable, automated, on-orbit microscope subrack facility, allowing flexible scheduling and control of fluids and biology experiments within the FIR. Key diagnostic capabilities for meeting science requirements include video microscopy to observe microscopic phenomena and dynamic interactions, interferometry to make thin film measurements with nanometer resolution, laser tweezers for particle manipulation, confocal microscopy to provide enhanced three-dimensional visualization of structures, and spectrophotometry to measure photonic properties of materials. The LMM also provides experiment sample containment for frangibles and fluids. This paper will provide a description of the current FIR and LMM designs, planned capabilities and key features. In addition a brief description of the initial five experiments planned for LMM/FIR will be provided.

The Pathologist 2.0: An Update on Digital Pathology in Veterinary Medicine.

PubMed

Bertram, Christof A; Klopfleisch, Robert

2017-09-01

Using light microscopy to describe the microarchitecture of normal and diseased tissues has changed very little since the middle of the 19th century. While the premise of histologic analysis remains intact, our relationship with the microscope is changing dramatically. Digital pathology offers new forms of visualization, and delivery of images is facilitated in unprecedented ways. This new technology can untether us entirely from our light microscopes, with many pathologists already performing their jobs using virtual microscopy. Several veterinary colleges have integrated virtual microscopy in their curriculum, and some diagnostic histopathology labs are switching to virtual microscopy as their main tool for the assessment of histologic specimens. Considering recent technical advancements of slide scanner and viewing software, digital pathology should now be considered a serious alternative to traditional light microscopy. This review therefore intends to give an overview of the current digital pathology technologies and their potential in all fields of veterinary pathology (ie, research, diagnostic service, and education). A future integration of digital pathology in the veterinary pathologist's workflow seems to be inevitable, and therefore it is proposed that trainees should be taught in digital pathology to keep up with the unavoidable digitization of the profession.

Light Microscopy Microscope Experiment

NASA Image and Video Library

2016-02-04

Ground testing for the first confocal Light Microscopy Microscope (LMM) Experiment. Procter and Gamble is working with NASA Glenn scientists to prepare for a study that examines product stabilizers in a microgravity environment. The particles in the tube glow orange because they have been fluorescently tagged with a dye that reacts to green laser lights to allow construction of a 3D image point by point. The experiment, which will be sent to the ISS later this year, will help P&G develop improved product stabilizers to extend shelf life and develop more environmentally friendly packaging.

Low-level laser therapy on MCF-7 cells: a micro-Fourier transform infrared spectroscopy study

NASA Astrophysics Data System (ADS)

Magrini, Taciana D.; dos Santos, Nathalia Villa; Milazzotto, Marcella Pecora; Cerchiaro, Giselle; da Silva Martinho, Herculano

2012-10-01

Low-level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably scale from photobiostimulation/photobioinhibition at the cellular level to the molecular level. The detailed mechanism underlying this effect remains unknown. This study quantifies some relevant aspects of LLLT related to molecular and cellular variations. Malignant breast cells (MCF-7) were exposed to spatially filtered light from a He-Ne laser (633 nm) with fluences of 5, 28.8, and 1000 mJ/cm2. The cell viability was evaluated by optical microscopy using the Trypan Blue viability test. The micro-Fourier transform infrared technique was employed to obtain the vibrational spectra of each experimental group (control and irradiated) and identify the relevant biochemical alterations that occurred due to the process. It was observed that the red light influenced the RNA, phosphate, and serine/threonine/tyrosine bands. We found that light can influence cell metabolism depending on the laser fluence. For 5 mJ/cm2, MCF-7 cells suffer bioinhibition with decreased metabolic rates. In contrast, for the 1 J/cm2 laser fluence, cells present biostimulation accompanied by a metabolic rate elevation. Surprisingly, at the intermediate fluence, 28.8 mJ/cm2, the metabolic rate is increased despite the absence of proliferative results. The data were interpreted within the retrograde signaling pathway mechanism activated with light irradiation.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

PubMed

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

2015-01-01

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Crystal morphology of sunflower wax in soybean oil organogel

USDA-ARS?s Scientific Manuscript database

While sunflower wax has been recognized as an excellent organogelator for edible oil, the detailed morphology of sunflower wax crystals formed in an edible oil organogel has not been fully understood. In this study, polarized light microscopy, phase contrast microscopy, scanning electron microscopy ...

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

DOE PAGES

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; ...

2016-01-01

Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy

PubMed Central

Shribak, Michael; Larkin, Kieran G.; Biggs, David

2017-01-01

Abstract. We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100×/1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ∼0.5  nm and lateral resolution if ∼300  nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems. PMID:28060991

Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

PubMed

Mukhamadiyarov, Rinat A; Sevostyanova, Victoria V; Shishkova, Daria K; Nokhrin, Andrey V; Sidorova, Olga D; Kutikhin, Anton G

2016-06-01

A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft. Copyright © 2016 Elsevier Ltd. All rights reserved.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.

PubMed

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

NASA Astrophysics Data System (ADS)

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Light sheet microscopy reveals more gradual light attenuation in light green versus dark green soybean leaves

USDA-ARS?s Scientific Manuscript database

Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This oversaturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light, causing a steep gradient in carbon fixation. Reducing chl content could create a mor...

Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

Performance of Local Light Microscopy and the ParaScreen Pan/Pf Rapid Diagnostic Test to Detect Malaria in Health Centers in Northwest Ethiopia

PubMed Central

Endeshaw, Tekola; Graves, Patricia M.; Ayele, Berhan; Mosher, Aryc W.; Gebre, Teshome; Ayalew, Firew; Genet, Asrat; Mesfin, Alemayehu; Shargie, Estifanos Biru; Tadesse, Zerihun; Teferi, Tesfaye; Melak, Berhanu; Richards, Frank O.; Emerson, Paul M.

2012-01-01

Background Diagnostic tests are recommended for suspected malaria cases before treatment, but comparative performance of microscopy and rapid diagnostic tests (RDTs) at rural health centers has rarely been studied compared to independent expert microscopy. Methods Participants (N = 1997) with presumptive malaria were recruited from ten health centers with a range of transmission intensities in Amhara Regional State, Northwest Ethiopia during October to December 2007. Microscopy and ParaScreen Pan/Pf® RDT were done immediately by health center technicians. Blood slides were re-examined later at a central laboratory by independent expert microscopists. Results Of 1,997 febrile patients, 475 (23.8%) were positive by expert microscopists, with 57.7% P.falciparum, 24.6% P.vivax and 17.7% mixed infections. Sensitivity of health center microscopists for any malaria species was >90% in five health centers (four of which had the highest prevalence), >70% in nine centers and 44% in one site with lowest prevalence. Specificity for health center microscopy was very good (>95%) in all centers. For ParaScreen RDT, sensitivity was ≥90% in three centers, ≥70% in six and <60% in four centers. Specificity was ≥90% in all centers except one where it was 85%. Conclusions Health center microscopists performed well in nine of the ten health centers; while for ParaScreen RDT they performed well in only six centers. Overall the accuracy of local microscopy exceeded that of RDT for all outcomes. This study supports the introduction of RDTs only if accompanied by appropriate training, frequent supervision and quality control at all levels. Deficiencies in RDT use at some health centers must be rectified before universal replacement of good routine microscopy with RDTs. Maintenance and strengthening of good quality microscopy remains a priority at health center level. PMID:22536317

Performance of local light microscopy and the ParaScreen Pan/Pf rapid diagnostic test to detect malaria in health centers in Northwest Ethiopia.

PubMed

Endeshaw, Tekola; Graves, Patricia M; Ayele, Berhan; Mosher, Aryc W; Gebre, Teshome; Ayalew, Firew; Genet, Asrat; Mesfin, Alemayehu; Shargie, Estifanos Biru; Tadesse, Zerihun; Teferi, Tesfaye; Melak, Berhanu; Richards, Frank O; Emerson, Paul M

2012-01-01

Diagnostic tests are recommended for suspected malaria cases before treatment, but comparative performance of microscopy and rapid diagnostic tests (RDTs) at rural health centers has rarely been studied compared to independent expert microscopy. Participants (N = 1997) with presumptive malaria were recruited from ten health centers with a range of transmission intensities in Amhara Regional State, Northwest Ethiopia during October to December 2007. Microscopy and ParaScreen Pan/Pf® RDT were done immediately by health center technicians. Blood slides were re-examined later at a central laboratory by independent expert microscopists. Of 1,997 febrile patients, 475 (23.8%) were positive by expert microscopists, with 57.7% P. falciparum, 24.6% P. vivax and 17.7% mixed infections. Sensitivity of health center microscopists for any malaria species was >90% in five health centers (four of which had the highest prevalence), >70% in nine centers and 44% in one site with lowest prevalence. Specificity for health center microscopy was very good (>95%) in all centers. For ParaScreen RDT, sensitivity was ≥90% in three centers, ≥70% in six and <60% in four centers. Specificity was ≥90% in all centers except one where it was 85%. Health center microscopists performed well in nine of the ten health centers; while for ParaScreen RDT they performed well in only six centers. Overall the accuracy of local microscopy exceeded that of RDT for all outcomes. This study supports the introduction of RDTs only if accompanied by appropriate training, frequent supervision and quality control at all levels. Deficiencies in RDT use at some health centers must be rectified before universal replacement of good routine microscopy with RDTs. Maintenance and strengthening of good quality microscopy remains a priority at health center level.

An electron microscopy examination of primary recrystallization in TD-nickel.

NASA Technical Reports Server (NTRS)

Petrovic, J. J.; Ebert, L. J.

1972-01-01

Primary recrystallization in TD-nickel 1 in. bar has previously been regarded as the process by which the initial fine grain structure is converted to a coarse grain size (increases in grain size by 500 times) under suitable deformation and annealing conditions. This process is dependent on deformation mode. While it occurs readily after rolling transverse to the bar axis and annealing (800 C), it is completely inhibited by longitudinal rolling and swaging deformations, even for very high (1320 C) annealing temperatures. A transmission electron microscopy examination of deformation and annealing substructures indicates that primary recrystallization in TD-nickel 1 in. bar actually occurs on the sub-light optical level, to produce a grain structure similar in size to the initial fine grained state.

Light-induced migration of retinal microglia into the subretinal space.

PubMed

Ng, T F; Streilein, J W

2001-12-01

To explore the effects of light exposure and deprivation on the distribution and function of microglia in the subretinal space of mice. Using a monoclonal antibody, 5D4, that identifies resting, ramified microglia, the distribution and density of microglia in the retina, and the subretinal space were determined by confocal microscopy and by immunohistochemistry of cryopreserved sections of eyes of albino and pigmented mice exposed to diverse levels of light, ranging from complete darkness to intense brightness. Axotomized retinal ganglion cells were retrograde labeled by fluorescent tracer to determine whether the marker colocalizes to 5D4+ cells. Electron microscopy was used to evaluate microglia for evidence of phagocytosis. 5D4+ microglia in pigmented eyes were limited to the inner retinal layers, but in albino eyes 5D4+ cells were found in the outer retinal layers and subretinal space as well. The subretinal space of eyes of albino mice raised from birth in complete darkness contained few 5D4+ cells, but exposure to light caused the rapid accumulation of 5D4+ cells at this site. 5D4+ cell density in the subretinal space correlated directly with intensity of ambient light. Retrograde labeling of axotomized ganglion cells resulted in 5D4+ cells in the subretinal space that contained the retrograde label. Subretinal microglia contained phagocytized rod outer segment discs. On intense light exposure, 5D4+ cells adopted an active morphology, but failed to express class II major histocompatibility complex (MHC) molecules. Light exposure induced retinal microglia migration into the subretinal space in albino mice. Subretinal microglia appeared to augment through phagocytosis the capacity of pigment epithelium to take up the photoreceptor debris of light toxicity. The unexpected presence of these cells in the subretinal space raises questions concerning their potential contribution to immune privilege in this space and to the fate of retinal transplants.

Quantitative phase imaging of human red blood cells using phase-shifting white light interference microscopy with colour fringe analysis

NASA Astrophysics Data System (ADS)

Singh Mehta, Dalip; Srivastava, Vishal

2012-11-01

We report quantitative phase imaging of human red blood cells (RBCs) using phase-shifting interference microscopy. Five phase-shifted white light interferograms are recorded using colour charge coupled device camera. White light interferograms were decomposed into red, green, and blue colour components. The phase-shifted interferograms of each colour were then processed by phase-shifting analysis and phase maps for red, green, and blue colours were reconstructed. Wavelength dependent refractive index profiles of RBCs were computed from the single set of white light interferogram. The present technique has great potential for non-invasive determination of refractive index variation and morphological features of cells and tissues.

High ferritin levels have major effects on the morphology of erythrocytes in Alzheimer's disease.

PubMed

Bester, Janette; Buys, Antoinette V; Lipinski, Boguslaw; Kell, Douglas B; Pretorius, Etheresia

2013-01-01

Unliganded iron both contributes to the pathology of Alzheimer's disease (AD) and also changes the morphology of erythrocytes (RBCs). We tested the hypothesis that these two facts might be linked, i.e., that the RBCs of AD individuals have a variant morphology, that might have diagnostic or prognostic value. We included a literature survey of AD and its relationships to the vascular system, followed by a laboratory study. Four different microscopy techniques were used and results statistically compared to analyze trends between high and normal serum ferritin (SF) AD individuals. Light and scanning electron microscopies showed little difference between the morphologies of RBCs taken from healthy individuals and from normal SF AD individuals. By contrast, there were substantial changes in the morphology of RBCs taken from high SF AD individuals. These differences were also observed using confocal microscopy and as a significantly greater membrane stiffness (measured using force-distance curves). We argue that high ferritin levels may contribute to an accelerated pathology in AD. Our findings reinforce the importance of (unliganded) iron in AD, and suggest the possibility both of an early diagnosis and some means of treating or slowing down the progress of this disease.

Resistance in mango against infection by Ceratocystis fimbriata.

PubMed

Araujo, Leonardo; Bispo, Wilka Messner Silva; Cacique, Isaías Severino; Moreira, Wiler Ribas; Rodrigues, Fabrício Ávila

2014-08-01

This study was designed to characterize and describe host cell responses of stem tissue to mango wilt disease caused by the fungus Ceratocystis fimbriata in Brazil. Disease progress was followed, through time, in inoculated stems for two cultivars, 'Ubá' (field resistant) and 'Haden' (field susceptible). Stem sections from inoculated areas were examined using fluorescence light microscopy and transmission and scanning electron microscopy, coupled with energy-dispersive X-ray microanalysis. Tissues from Ubá colonized by C. fimbriata had stronger autofluorescence than those from Haden. The X-ray microanalysis revealed that the tissues of Ubá had higher levels of insoluble sulfur and calcium than those of Haden. Scanning electron microscopy revealed that fungal hyphae, chlamydospores (aleurioconidia), and perithecia-like structures of C. fimbriata were more abundant in Haden relative to Ubá. At the ultrastructural level, pathogen hyphae had grown into the degraded walls of parenchyma, fiber cells, and xylem vessels in the tissue of Haden. However, in Ubá, plant cell walls were rarely degraded and hyphae were often surrounded by dense, amorphous granular materials and hyphae appeared to have died. Taken together, the results of this study characterize the susceptible and resistant basal cell responses of mango stem tissue to infection by C. fimbriata.

Prevalence of Malaria in Pregnant Women in Lagos, South-West Nigeria

PubMed Central

Agomo, Chimere O.; Anorlu, Rose I.; Agomo, Philip U.

2009-01-01

Prevalence rates reported for malaria in pregnancy in Nigeria vary considerably. The accuracy of results of malaria diagnosis is dependent on training, experience, and motivation of the microscopist as well as the laboratory facility available. Results of training programmes on malaria microscopy have shown low levels of sensitivity and specificity of those involved in malaria diagnosis routinely and for research. This study was done to ascertain the true prevalence of malaria in pregnancy in Lagos, South-West Nigeria. A total of 1,084 pregnant women were recruited into this study. Blood smears stained with Giemsa were used for malaria diagnosis by light microscopy. Malaria infection during pregnancy presents mostly as asymptomatic infection. The prevalence of malaria in this population was 7.7% (95% confidence interval; 6.2-9.4%). Factors identified to increase the risk of malaria infection include young maternal age (< 20 years), and gravidity (primigravida). In conclusion, this study exposes the over-diagnosis of malaria in pregnancy and the need for training and retraining of laboratory staffs as well as establishing the malaria diagnosis quality assurance programme to ensure the accuracy of malaria microscopy results at all levels. PMID:19488427

Impact of high hydrostatic pressure and pasteurization on the structure and the extractability of bioactive compounds of persimmon “Rojo Brillante”.

PubMed

Hernández-Carrión, M; Vázquez-Gutiérrez, J L; Hernando, I; Quiles, A

2014-01-01

Rojo Brillante is an astringent oriental persimmon variety with high levels of bioactive compounds such as soluble tannins, carotenoids, phenolic acids, and dietary fiber. The purpose of this study was to investigate the effects of high hydrostatic pressure (HHP) and pasteurization on the structure of the fruit and on the extractability of certain bioactive compounds. The microstructure was studied using light microscopy, transmission electron microscopy, and low temperature scanning electron microscopy, and certain physicochemical properties (carotenoid and total soluble tannin content, antioxidant activity, fiber content, color, and texture properties) were measured. The structural changes induced by HHP caused a rise in solute circulation in the tissues that could be responsible for the increased carotenoid level and the unchanged antioxidant activity in comparison with the untreated persimmon. In contrast, the changes that took place during pasteurization lowered the tannin content and antioxidant activity. Consequently, HHP treatment could improve the extraction of potentially bioactive compoundsxsts from persimmons. A high nutritional value ingredient to be used when formulating new functional foods could be obtained using HHP. © 2013 Institute of Food Technologists®

Characterisation of a major phytoplankton bloom in the River Thames (UK) using flow cytometry and high performance liquid chromatography.

PubMed

Moorhouse, H L; Read, D S; McGowan, S; Wagner, M; Roberts, C; Armstrong, L K; Nicholls, D J E; Wickham, H D; Hutchins, M G; Bowes, M J

2018-05-15

Recent river studies have observed rapid phytoplankton dynamics, driven by diurnal cycling and short-term responses to storm events, highlighting the need to adopt new high-frequency characterisation methods to understand these complex ecological systems. This study utilised two such analytical methods; pigment analysis by high performance liquid chromatography (HPLC) and cell counting by flow cytometry (FCM), alongside traditional chlorophyll spectrophotometry and light microscopy screening, to characterise the major phytoplankton bloom of 2015 in the River Thames, UK. All analytical techniques observed a rapid increase in chlorophyll a concentration and cell abundances from March to early June, caused primarily by a diatom bloom. Light microscopy identified a shift from pennate to centric diatoms during this period. The initial diatom bloom coincided with increased HPLC peridinin concentrations, indicating the presence of dinoflagellates which were likely to be consuming the diatom population. The diatom bloom declined rapidly in early June, coinciding with a storm event. There were low chlorophyll a concentrations (by both HPLC and spectrophotometric methods) throughout July and August, implying low biomass and phytoplankton activity. However, FCM revealed high abundances of pico-chlorophytes and cyanobacteria through July and August, showing that phytoplankton communities remain active and abundant throughout the summer period. In combination, these techniques are able to simultaneously characterise a wider range of phytoplankton groups, with greater certainty, and provide improved understanding of phytoplankton functioning (e.g. production of UV inhibiting pigments by cyanobacteria in response to high light levels) and ecological status (through examination of pigment degradation products). Combined HPLC and FCM analyses offer rapid and cost-effective characterisation of phytoplankton communities at appropriate timescales. This will allow a more-targeted use of light microscopy to capture phytoplankton peaks or to investigate periods of rapid community succession. This will lead to greater system understanding of phytoplankton succession in response to biogeochemical drivers. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout, and first description of erythrophore ultrastructure in salmonids

PubMed Central

Djurdjevič, Ida; Kreft, Mateja Erdani; Sušnik Bajec, Simona

2015-01-01

Skin pigmentation in animals is an important trait with many functions. The present study focused on two closely related salmonid species, marble trout (Salmo marmoratus) and brown trout (S. trutta), which display an uncommon labyrinthine (marble-like) and spot skin pattern, respectively. To determine the role of chromatophore type in the different formation of skin pigment patterns in the two species, the distribution and ultrastructure of chromatophores was examined with light microscopy and transmission electron microscopy. The presence of three types of chromatophores in trout skin was confirmed: melanophores; xanthophores; and iridophores. In addition, using correlative microscopy, erythrophore ultrastructure in salmonids was described for the first time. Two types of erythrophores are distinguished, both located exclusively in the skin of brown trout: type 1 in black spot skin sections similar to xanthophores; and type 2 with a unique ultrastructure, located only in red spot skin sections. Morphologically, the difference between the light and dark pigmentation of trout skin depends primarily on the position and density of melanophores, in the dark region covering other chromatophores, and in the light region with the iridophores and xanthophores usually exposed. With larger amounts of melanophores, absence of xanthophores and presence of erythrophores type 1 and type L iridophores in the black spot compared with the light regions and the presence of erythrophores type 2 in the red spot, a higher level of pigment cell organisation in the skin of brown trout compared with that of marble trout was demonstrated. Even though the skin regions with chromatophores were well defined, not all the chromatophores were in direct contact, either homophilically or heterophilically, with each other. In addition to short-range interactions, an important role of the cellular environment and long-range interactions between chromatophores in promoting adult pigment pattern formation of trout are proposed. PMID:26467239

Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout, and first description of erythrophore ultrastructure in salmonids.

PubMed

Djurdjevič, Ida; Kreft, Mateja Erdani; Sušnik Bajec, Simona

2015-11-01

Skin pigmentation in animals is an important trait with many functions. The present study focused on two closely related salmonid species, marble trout (Salmo marmoratus) and brown trout (S. trutta), which display an uncommon labyrinthine (marble-like) and spot skin pattern, respectively. To determine the role of chromatophore type in the different formation of skin pigment patterns in the two species, the distribution and ultrastructure of chromatophores was examined with light microscopy and transmission electron microscopy. The presence of three types of chromatophores in trout skin was confirmed: melanophores; xanthophores; and iridophores. In addition, using correlative microscopy, erythrophore ultrastructure in salmonids was described for the first time. Two types of erythrophores are distinguished, both located exclusively in the skin of brown trout: type 1 in black spot skin sections similar to xanthophores; and type 2 with a unique ultrastructure, located only in red spot skin sections. Morphologically, the difference between the light and dark pigmentation of trout skin depends primarily on the position and density of melanophores, in the dark region covering other chromatophores, and in the light region with the iridophores and xanthophores usually exposed. With larger amounts of melanophores, absence of xanthophores and presence of erythrophores type 1 and type L iridophores in the black spot compared with the light regions and the presence of erythrophores type 2 in the red spot, a higher level of pigment cell organisation in the skin of brown trout compared with that of marble trout was demonstrated. Even though the skin regions with chromatophores were well defined, not all the chromatophores were in direct contact, either homophilically or heterophilically, with each other. In addition to short-range interactions, an important role of the cellular environment and long-range interactions between chromatophores in promoting adult pigment pattern formation of trout are proposed. © 2015 Anatomical Society.

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Application of Multiphoton Microscopy in Dermatological Studies: a Mini-Review

PubMed Central

Yew, Elijah; Rowlands, Christopher

2014-01-01

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance. PMID:25075226

Imaging cellular and subcellular structure of human brain tissue using micro computed tomography

NASA Astrophysics Data System (ADS)

Khimchenko, Anna; Bikis, Christos; Schweighauser, Gabriel; Hench, Jürgen; Joita-Pacureanu, Alexandra-Teodora; Thalmann, Peter; Deyhle, Hans; Osmani, Bekim; Chicherova, Natalia; Hieber, Simone E.; Cloetens, Peter; Müller-Gerbl, Magdalena; Schulz, Georg; Müller, Bert

2017-09-01

Brain tissues have been an attractive subject for investigations in neuropathology, neuroscience, and neurobiol- ogy. Nevertheless, existing imaging methodologies have intrinsic limitations in three-dimensional (3D) label-free visualisation of extended tissue samples down to (sub)cellular level. For a long time, these morphological features were visualised by electron or light microscopies. In addition to being time-consuming, microscopic investigation includes specimen fixation, embedding, sectioning, staining, and imaging with the associated artefacts. More- over, optical microscopy remains hampered by a fundamental limit in the spatial resolution that is imposed by the diffraction of visible light wavefront. In contrast, various tomography approaches do not require a complex specimen preparation and can now reach a true (sub)cellular resolution. Even laboratory-based micro computed tomography in the absorption-contrast mode of formalin-fixed paraffin-embedded (FFPE) human cerebellum yields an image contrast comparable to conventional histological sections. Data of a superior image quality was obtained by means of synchrotron radiation-based single-distance X-ray phase-contrast tomography enabling the visualisation of non-stained Purkinje cells down to the subcellular level and automated cell counting. The question arises, whether the data quality of the hard X-ray tomography can be superior to optical microscopy. Herein, we discuss the label-free investigation of the human brain ultramorphology be means of synchrotron radiation-based hard X-ray magnified phase-contrast in-line tomography at the nano-imaging beamline ID16A (ESRF, Grenoble, France). As an example, we present images of FFPE human cerebellum block. Hard X-ray tomography can provide detailed information on human tissues in health and disease with a spatial resolution below the optical limit, improving understanding of the neuro-degenerative diseases.

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

PubMed Central

Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Shibue, Tsukasa; Schwab, Yannick; Goetz, Jacky G.

2014-01-01

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. PMID:25479106

Ultrastructural study on Biomphalaria alexandrina haemocytes infected with Schistosoma mansoni in Egypt and its correlation with nitric oxide level.

PubMed

Helal, Eman G; El-Dafrawy, Shadia M; Mohamed, Amira H; Abou-El-Nour, Basma M; Ibrahim, Samah

2014-04-01

Some snails of Biomphalaria alexandrina can resist the infection of Schistosoma mansoni so this study aimed to clearly this mechanism by using light and electron microscopy (EM) and determine the role of Nitric oxide in this mechanism. B. alexandrina snails used in this study were exposed individually to S. mansoni infection according to their response they were classified into susceptible group (shed cercariae) and resistant group (failed to shed cercariae). Snails not exposed to infection were included in this study as control group. Nitric oxide (NO) level was assayed directly in the soluble fraction of B. alexandrina haemolymph supernatants collected from each group of B. alexandrina snails were subjected to NO assay by the Greiss reaction. The level of NO in haemolymph of infected snails was significantly increased (p < 0.001) than both control and non infected snails groups, however, in non infected snails group had significantly (p < 0.05) compared to control group. This study when correlated the changes recognized by EM with NO level the pro apoptotic effect of high level of NO on the haemocytes. Characterization and identification of cell shape of haemocytes in both haemolymph and tissue were examined by light and electron microscopy. Examination of B. alexandrina snail's haemocytes revealed three types of different cells classified according to their shape and granular contents. These cells are granulocytes, amoebocytes and hyalineocytes. Electron microscope study also revealed the important role of granulocytes and amoebocytes as defense mechanism against snail infection. NO is considered an important anti parasite molecule; intra-molluscan stages of parasites switch off host NO defense response.

Correlative Instrumental Neutron Activation Analysis, Light Microscopy, Transmission Electron Microscopy, and X-ray Microanalysis for Qualitative and Quantitative Detection of Colloidal Gold Spheres in Biological Specimens

NASA Astrophysics Data System (ADS)

Hillyer, Julián F.; Albrecht, Ralph M.

1998-10-01

: Colloidal gold, conjugated to ligands or antibodies, is routinely used as a label for the detection of cell structures by light (LM) and electron microscopy (EM). To date, several methods to count the number of colloidal gold labels have been employed with limited success. Instrumental neutron activation analysis (INAA), a physical method for the analysis of the elemental composition of materials, can be used to provide a quantitative index of gold accumulation in bulk specimens. Given that gold is not naturally found in biological specimens in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be calculated in bulk biological samples by INAA. Here we describe the use of INAA, LM, transmission EM, and X-ray microanalysis (EDX) in a model to determine both distribution (localization) and amount of colloidal gold at the organ, tissue, cellular, and ultrastructural levels in whole animal systems following administration. In addition, the sensitivity for gold in biological specimens by INAA is compared with that of inductively coupled plasma mass spectrometry (ICP-MS). The correlative use of INAA, LM, TEM, and EDX can be useful, for example, in the quantitative and qualitative tracking of various labeled molecular species following administration in vivo.

Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

NASA Astrophysics Data System (ADS)

Wong, Terence T. W.; Zhang, Ruiying; Hsu, Hsun-Chia; Maslov, Konstantin I.; Shi, Junhui; Chen, Ruimin; Shung, K. Kirk; Zhou, Qifa; Wang, Lihong V.

2018-02-01

In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

High-Contrast Imaging of Cholesterol Crystals in Rabbit Arteries Ex Vivo Using LED-Based Polarization Microscopy.

PubMed

Cho, Seonghee; Kim, Kyungmin; Kim, Taehoon; Park, Hyoeun; Kim, Jin-Moo; Lee, SeungHoon; Kang, YeonSu; Chang, Kiyuk; Kim, Chulhong

2018-04-19

Detection of cholesterol crystals (Chcs) in atherosclerosis disease is important for understanding the pathophysiology of atherosclerosis. Polarization microscopy (PM) has been in use traditionally for detecting Chcs, but they have difficulty in distinguishing Chcs with other crystalline materials in tissue, such as collagens. Thus, most studies using PM have been limited to studying cell-level samples. Although various methods have been proposed to detect Chcs with high specificity, most of them have low signal-to-noise ratios, a high system construction cost, and are difficult to operate due to a complex protocol. To address these problems, we have developed a simple and inexpensive universal serial bus (USB) PM system equipped with a 5700 K cool-white light-emitting diode (LED). In this system, Chcs are shown in a light blue color while collagen is shown in a yellow color. More importantly, the contrast between Chcs and collagens is improved by a factor of 2.3 under an aqueous condition in these PM images. These imaging results are well-matched with the ones acquired with two-photon microscopy (TPM). The system can visualize the features of atherosclerosis that cannot be visualized by the conventional hematoxylin and eosin and oil-red-o staining methods. Thus, we believe that this simple USB PM system can be widely used to identify Chcs in atherosclerosis.

[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction].

PubMed

González-Ortega, C; Cancino-Villarreal, P; Alonzo-Torres, V E; Martínez-Robles, I; Pérez-Peña, E; Gutiérrez-Gutiérrez, A M

2016-04-01

Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

Ag3PO4 nanocrystals deposited on monoclinic olive-like BiVO4 with efficient photodegradation of organic dyes under visible light irradiation

NASA Astrophysics Data System (ADS)

Chen, Jingshuai; Jiang, Liang-Liang; Liu, Xing-Pei; Mao, Chang-Jie; Song, Ji-Ming; Niu, Helin; Zhang, Shengyi

2017-05-01

Olive-like BiVO4 microstructures with lengths of 600-1000 nm and widths of 300-600 nm have been synthesized via a facile and additive-free solvothermal method. Studies find that the type of solvent plays an important role in the morphology of the final products. Furthermore, Ag3PO4 nanocrystals are successfully deposited on monoclinic olive-like BiVO4 via in situ precipitation method. The as-synthesized samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), photoluminescence (PL) spectra, and UV-vis diffuse reflectance spectra (DRS). The photocatalytic activities of the catalysts are evaluated by degradation of rhodamine B (RhB) and methylene blue (MB) under visible light (≥420 nm) irradiation. The experimental results suggest that catalytic activity of the composite photocatalysts is greatly influenced by the loading level of Ag3PO4. The molar ratio of 0.8:1.0 Ag3PO4-loaded BiVO4 exhibits higher photocatalytic activity in both the decolorization of RhB and MB than that of individual BiVO4 and P25. The observed improvement in photocatalytic activity is associated with the extended absorption in the visible light region resulting from the Ag3PO4 nanoparticles, and the effective separation of photogenerated carriers at the Ag3PO4/BiVO4 interfaces through the formation of heterojunction structure. The study provides a general and effective method in the fabrication of composite with sound heterojunctions that may show a variety of applications.

Direct correlations of structural and optical properties of three-dimensional GaN/InGaN core/shell micro-light emitting diodes

NASA Astrophysics Data System (ADS)

Sadat Mohajerani, Matin; Müller, Marcus; Hartmann, Jana; Zhou, Hao; Wehmann, Hergo-H.; Veit, Peter; Bertram, Frank; Christen, Jürgen; Waag, Andreas

2016-05-01

Three-dimensional (3D) InGaN/GaN quantum-well (QW) core-shell light emitting diodes (LEDs) are a promising candidate for the future solid state lighting. In this contribution, we study direct correlations of structural and optical properties of the core-shell LEDs using highly spatially-resolved cathodoluminescence spectroscopy (CL) in combination with scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM). Temperature-dependent resonant photoluminescence (PL) spectroscopy has been performed to understand recombination mechanisms and to estimate the internal quantum efficiency (IQE).

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

PubMed

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Advanced SLMs for microscopy

NASA Astrophysics Data System (ADS)

Linnenberger, A.

2018-02-01

Wavefront shaping devices such as deformable mirrors, liquid crystal spatial light modulators (SLMs), and active lenses are of considerable interest in microscopy for aberration correction, volumetric imaging, and programmable excitation. Liquid crystal SLMs are high resolution phase modulators capable of creating complex phase profiles to reshape, or redirect light within a three-dimensional (3D) volume. Recent advances in Meadowlark Optics (MLO) SLMs reduce losses by increasing fill factor from 83.4% to 96%, and improving resolution from 512 x 512 pixels to 1920 x 1152 pixels while maintaining a liquid crystal response time of 300 Hz at 1064 nm. This paper summarizes new SLM capabilities, and benefits for microscopy.

Pterygodermatites (Mesopectines) quentini (Nematoda, Rictulariidae), a parasite of Praomys rostratus (Rodentia, Muridae) in Mali: scanning electron and light microscopy

PubMed Central

2013-01-01

Pterygodermatites (Mesopectines) quentini n. sp. (Nematoda, Rictulariidae) is described from the murine host Praomys rostratus in the south of the Republic of Mali. It differs from other species of the subgenus by the morphology of the head, which bears four simple cephalic papillae and a nearly axial oral opening, the number of caudal papillae, the number of precloacal cuticular formations, unequal spicules and the ratio of spicule lengths/body length. The use of scanning electron microscopy in combination with conventional light microscopy enabled us to give a detailed description of the morphological characters of this new species. PMID:24025692

Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy.

PubMed

Ryan, Duncan P; Gould, Elizabeth A; Seedorf, Gregory J; Masihzadeh, Omid; Abman, Steven H; Vijayaraghavan, Sukumar; Macklin, Wendy B; Restrepo, Diego; Shepherd, Douglas P

2017-09-20

Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

PubMed Central

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast. PMID:20210471

Calibrating excitation light fluxes for quantitative light microscopy in cell biology

PubMed Central

Grünwald, David; Shenoy, Shailesh M; Burke, Sean; Singer, Robert H

2011-01-01

Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp. PMID:18974739

Radial super-resolution in digital holographic microscopy using structured illumination with circular symmetry

NASA Astrophysics Data System (ADS)

Yin, Yujian; Su, Ping; Ma, Jianshe

2018-01-01

A method to improve the radial resolution using special structured light is proposed in the field of digital holographic microscopy (DHM). A specimen is illuminated with circular symmetrical structured light that makes the spectrum have radial movement, so that high frequency components of the specimen are moved into the passband of the receiver to overcome the diffraction limit. In the DHM imaging system, Computer Generated Hologram (CGH) technology is used to generate the required structured light grating. Then the grating is loaded into a spatial light modulator (SLM) to obtain specific structured illumination. After recording the hologram, digital reconstruction, for the microstructure of a binary optical element that needs to observe radial distribution, the radial resolution of the specimen is improved experimentally compare it with the result of one-dimensional sinusoidal structured light imaging. And a method of designing structured light is presented.

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy, morphometry and confocal laser scanning microscopy.

PubMed

Souza, Joyce; Garcia, Juberlan; Neves, Renata H; Machado-Silva, José Roberto; Maldonado, Arnaldo

2013-12-01

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy. Copyright © 2013. Published by Elsevier Inc.

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

PubMed Central

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

Microstructure of Dense Thin Sheets of gamma-TiAl Fabricated by Hot Isostatic Pressing of Tape-Cast Monotapes (Preprint)

DTIC Science & Technology

2007-02-01

fabrication of dense thin sheets of gamma titanium aluminide . Polarized light microscopy revealed a fine-grained microstructure but a few isolated...HIPed (near-gamma) microstructure occurred. 15. SUBJECT TERMS gamma titanium aluminide , thin sheet, tape casting, hot isostatic pressing 16...sheets (250–300 μm thick) of gamma titanium aluminide (γ-TiAl). Polarized light microscopy revealed a fine-grained microstructure (average grain

Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll f.

PubMed

Majumder, Erica L-W; Wolf, Benjamin M; Liu, Haijun; Berg, R Howard; Timlin, Jerilyn A; Chen, Min; Blankenship, Robert E

2017-11-01

Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. In this study, cyanobacteria containing chlorophyll f, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris, were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl f was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

Advances in Light Microscopy for Neuroscience

PubMed Central

Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

2010-01-01

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

Analysis of Cutmarks on Bone: Can Light Microscopy Be of Any Help?

PubMed

Cerutti, Elisa; Spagnoli, Laura; Araujo, Nadezhda; Gibelli, Daniele; Mazzarelli, Debora; Cattaneo, Cristina

2016-12-01

One of the main issues in forensic anthropology consists of the identification of signs of trauma in skeletal remains, including sharp-force injuries. So far, several studies have been performed to assess differences between injuries caused by different instruments, not, however, through light microscopy.In this study, 152 sharp-force injuries were performed by 5 different tools through 2 different orientations on 2 humeral diaphyses and were analyzed by stereo and light microscopy to assess possible morphological differences.This study showed that although W-shaped injuries are frequently reported in cases of wood-cutting saws, other shapes are often observed; lesions due to metal-cutting saws are almost always U shaped, whereas injuries caused by knives are V shaped. Although cut marks may represent a variable range of features, the present study was able to highlight typical profiles that may be of some help for the diagnosis of weapon and the intentionality of the action.

Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

NASA Astrophysics Data System (ADS)

Cremer, Christoph; Birk, Udo

2016-04-01

The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

Phase Sensitive Demodulation in Multiphoton Microscopy

NASA Astrophysics Data System (ADS)

Fisher, Walt G.; Piston, David W.; Wachter, Eric A.

2002-06-01

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

Multispectral digital lensless holographic microscopy: from femtosecond laser to white light LED

NASA Astrophysics Data System (ADS)

Garcia-Sucerquia, J.

2015-04-01

The use of femtosecond laser radiation and super bright white LED in digital lensless holographic microscopy is presented. For the ultrafast laser radiation two different configurations of operation of the microscope are presented and the dissimilar performance of each one analyzed. The microscope operating with a super bright white light LED in combination with optical filters shows very competitive performance as it is compared with more expensive optical sources. The broadband emission of both radiation sources allows the multispectral imaging of biological samples to obtain spectral responses and/or full color images of the microscopic specimens; sections of the head of a Drosophila melanogaster fly are imaged in this contribution. The simple, solid, compact, lightweight, and reliable architecture of digital lensless holographic microscopy operating with broadband light sources to image biological specimens exhibiting micrometer-sized details is evaluated in the present contribution.

Visible light alters yeast metabolic rhythms by inhibiting respiration.

PubMed

Robertson, James Brian; Davis, Chris R; Johnson, Carl Hirschie

2013-12-24

Exposure of cells to visible light in nature or in fluorescence microscopy often is considered to be relatively innocuous. However, using the yeast respiratory oscillation (YRO) as a sensitive measurement of metabolism, we find that non-UV visible light has a significant impact on yeast metabolism. Blue/green wavelengths of visible light shorten the period and dampen the amplitude of the YRO, which is an ultradian rhythm of cell metabolism and transcription. The wavelengths of light that have the greatest effect coincide with the peak absorption regions of cytochromes. Moreover, treating yeast with the electron transport inhibitor sodium azide has similar effects on the YRO as visible light. Because impairment of respiration by light would change several state variables believed to play vital roles in the YRO (e.g., oxygen tension and ATP levels), we tested oxygen's role in YRO stability and found that externally induced oxygen depletion can reset the phase of the oscillation, demonstrating that respiratory capacity plays a role in the oscillation's period and phase. Light-induced damage to the cytochromes also produces reactive oxygen species that up-regulate the oxidative stress response gene TRX2 that is involved in pathways that enable sustained growth in bright visible light. Therefore, visible light can modulate cellular rhythmicity and metabolism through unexpectedly photosensitive pathways.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

PubMed

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

PubMed

Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

2015-08-01

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

An Investigation of the Incorporation of Virtual Microscopy in the Cytotechnology Educational Curriculum

ERIC Educational Resources Information Center

Mukherjee, Maheswari S.

2012-01-01

Traditionally, cytotechnology (CT) students have been trained by using light microscopy (LM) and glass slides. However, this method of training has some drawbacks. Several other educational programs with similar issues have incorporated virtual microscopy (VM) in their curricula. In VM, the specimens on glass slides are converted into virtual…

Scanning Capacitance Microscopy | Materials Science | NREL

Science.gov Websites

obtained using scanning capacitance microscopy. Top Right: Image of p-type and n-type material, obtained 'fingers' of light-colored n-type material on a yellow and blue background representing p-type material ; measurement data were obtained using scanning capacitance microscopy. Bottom Right: Image of p-type and n-type

Electronic Blending in Virtual Microscopy

ERIC Educational Resources Information Center

Maybury, Terrence S.; Farah, Camile S.

2010-01-01

Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

A national facility for biological cryo-electron microscopy

PubMed Central

Saibil, Helen R.; Grünewald, Kay; Stuart, David I.

2015-01-01

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback. PMID:25615867

The coming paradigm shift: A transition from manual to automated microscopy.

PubMed

Farahani, Navid; Monteith, Corey E

2016-01-01

The field of pathology has used light microscopy (LM) extensively since the mid-19(th) century for examination of histological tissue preparations. This technology has remained the foremost tool in use by pathologists even as other fields have undergone a great change in recent years through new technologies. However, as new microscopy techniques are perfected and made available, this reliance on the standard LM will likely begin to change. Advanced imaging involving both diffraction-limited and subdiffraction techniques are bringing nondestructive, high-resolution, molecular-level imaging to pathology. Some of these technologies can produce three-dimensional (3D) datasets from sampled tissues. In addition, block-face/tissue-sectioning techniques are already providing automated, large-scale 3D datasets of whole specimens. These datasets allow pathologists to see an entire sample with all of its spatial information intact, and furthermore allow image analysis such as detection, segmentation, and classification, which are impossible in standard LM. It is likely that these technologies herald a major paradigm shift in the field of pathology.

Structural and Ultrastructural Characteristics of Bone-Tendon Junction of the Calcaneal Tendon of Adult and Elderly Wistar Rats

PubMed Central

Cury, Diego Pulzatto; Dias, Fernando José; Miglino, Maria Angélica; Watanabe, Ii-sei

2016-01-01

Tendons are transition tissues that transfer the contractile forces generated by the muscles to the bones, allowing movement. The region where the tendon attaches to the bone is called bone-tendon junction or enthesis and may be classified as fibrous or fibrocartilaginous. This study aims to analyze the collagen fibers and the cells present in the bone-tendon junction using light microscopy and ultrastructural techniques as scanning electron microscopy and transmission electron microscopy. Forty male Wistar rats were used in the experiment, being 20 adult rats at 4 months-old and 20 elderly rats at 20 months-old. The hind limbs of the rats were removed, dissected and prepared to light microscopy, transmission electron microscopy and scanning electron microscopy. The aging process showed changes in the collagen fibrils, with a predominance of type III fibers in the elderly group, in addition to a decrease in the amount of the fibrocartilage cells, fewer and shorter cytoplasmic processes and a decreased synthetic capacity due to degradation of the organelles involved in synthesis. PMID:27078690

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

PubMed Central

HÖHN, K.; FUCHS, J.; FRÖBER, A.; KIRMSE, R.; GLASS, B.; ANDERS‐ÖSSWEIN, M.; WALTHER, P.; KRÄUSSLICH, H.‐G.

2015-01-01

Summary In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells. PMID:25786567

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Structural and Ultrastructural Characteristics of Bone-Tendon Junction of the Calcaneal Tendon of Adult and Elderly Wistar Rats.

PubMed

Cury, Diego Pulzatto; Dias, Fernando José; Miglino, Maria Angélica; Watanabe, Ii-sei

2016-01-01

Tendons are transition tissues that transfer the contractile forces generated by the muscles to the bones, allowing movement. The region where the tendon attaches to the bone is called bone-tendon junction or enthesis and may be classified as fibrous or fibrocartilaginous. This study aims to analyze the collagen fibers and the cells present in the bone-tendon junction using light microscopy and ultrastructural techniques as scanning electron microscopy and transmission electron microscopy. Forty male Wistar rats were used in the experiment, being 20 adult rats at 4 months-old and 20 elderly rats at 20 months-old. The hind limbs of the rats were removed, dissected and prepared to light microscopy, transmission electron microscopy and scanning electron microscopy. The aging process showed changes in the collagen fibrils, with a predominance of type III fibers in the elderly group, in addition to a decrease in the amount of the fibrocartilage cells, fewer and shorter cytoplasmic processes and a decreased synthetic capacity due to degradation of the organelles involved in synthesis.

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

PubMed Central

Chiu, L-D; Su, L; Reichelt, S; Amos, WB

2012-01-01

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

Acute unclassified leukemia: A clinicopathologic study with diagnostic implications of electron microscopy.

PubMed

Youness, E; Trujillo, J M; Ahearn, M J; McCredie, K B; Cork, A

1980-01-01

By rigid cytological and cytochemical criteria, the diagnosis of acute and undifferentiated leukemia was established in 22 patients. According to defined criteria, the leukemic cells could not be classified by conventional light microscopic techniques employed in the study of hematopoietic tissue. Cytochemical studies including peroxidase, periodic acid schiff (PAS) and nonspecific esterase (alpha napthyl butyrate-reacting esterase) stains were done on fresh bone marrow samples, and the percentage of positive leukemia cells for each of these stains was determined on 200 cells. In this series of leukemias, cytochemistry at the light microscope level did not contribute to further classification. Subsequent electron microscopic examination of bone marrow samples from these patients confirmed the immaturity and nuclear/cytoplasmic asynchrony of the leukemic cells. Several in vivo neoplastic markers, such as nuclear blebs, increased nuclear bodies, and cytoplasmic fibrillar bundles could be demonstrated in these cells. Fourteen cases from this series exhibited peroxidase-positive developmental granule formation at the ultrastructural level and were reclassified as acute granulocyte leukemia (AGL). One case was reclassified as lymphoma (poor differentiated type), one case was diagnosed as acute monocytic leukemia (AmonoL), and six cases remained in the undifferentiated category (AUL). Clinical and laboratory features, response to treatment, and survival data were evaluated for these patients. This study demonstrated that electron microscopy is useful in the cytological diagnosis of human leukemia.

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

Setting up and running an advanced light microscopy and imaging facility.

PubMed

Sánchez, Carlos; Muñoz, Ma Ángeles; Villalba, Maite; Labrador, Verónica; Díez-Guerra, F Javier

2011-07-01

During the last twenty years, interest in light microscopy and imaging techniques has grown in various fields, such as molecular and cellular biology, developmental biology, and neurobiology. In addition, the number of scientific articles and journals using these techniques is rapidly increasing. Nowadays, most research institutions require sophisticated microscopy systems to cover their investigation demands. In general, such instruments are too expensive and complex to be purchased and managed by a single laboratory or research group, so they have to be shared with other groups and supervised by specialized personnel. This is the reason why microscopy and imaging facilities are becoming so important at research institutions nowadays. In this unit, we have gathered and presented a number of issues and considerations from our own experience that we hope will be helpful when planning or setting up a new facility.

Visualization of HIV T Cell Virological Synapses and Virus-Containing Compartments by Three-Dimensional Correlative Light and Electron Microscopy

PubMed Central

Wang, Lili; Eng, Edward T.; Law, Kenneth; Gordon, Ronald E.; Rice, William J.

2016-01-01

ABSTRACT Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses. PMID:27847357

Optimization of the excitation light sheet in selective plane illumination microscopy

PubMed Central

Gao, Liang

2015-01-01

Selective plane illumination microscopy (SPIM) allows rapid 3D live fluorescence imaging on biological specimens with high 3D spatial resolution, good optical sectioning capability and minimal photobleaching and phototoxic effect. SPIM gains its advantage by confining the excitation light near the detection focal plane, and its performance is determined by the ability to create a thin, large and uniform excitation light sheet. Several methods have been developed to create such an excitation light sheet for SPIM. However, each method has its own strengths and weaknesses, and tradeoffs must be made among different aspects in SPIM imaging. In this work, we present a strategy to select the excitation light sheet among the latest SPIM techniques, and to optimize its geometry based on spatial resolution, field of view, optical sectioning capability, and the sample to be imaged. Besides the light sheets discussed in this work, the proposed strategy is also applicable to estimate the SPIM performance using other excitation light sheets. PMID:25798312

Vasotropic light-chain amyloidosis and ischaemic cholangiopathy.

PubMed

Johnston, Emma L; Wilkinson, Mark; Knisely, A S

2015-06-25

A 75-year-old woman was incidentally found to have deranged liver function tests (LFTs). She was well, apart from 2 years of dyspnoea. Investigations had revealed atrial fibrillation and a right pleural effusion, without identified aetiology. On examination, the only finding was a palpable liver edge. Initial blood and ultrasound screening suggested no cause. The patient underwent liver biopsy. Microscopy showed κ-immunoglobulin light chains deposited exclusively in portal tracts, within blood vessel and bile duct walls. This pattern, although unusual, raised the possibility of κ-light chain disease. Serum electrophoresis was normal, as were serum immunoglobulin values. Serum concentrations of κ-light chains were elevated and microscopy of aspirated bone marrow found light-chain deposits with 10% plasmacytosis. Serum amyloid P (SAP) scintigraphy demonstrated splenic uptake. Myeloma, κ-light chain, with light-chain amyloidosis was diagnosed. The patient has responded well to cyclophosphamide, bortazomib and dexamethasone chemotherapy, and her LFTs are now nearly normal. 2015 BMJ Publishing Group Ltd.

Fractal propagation method enables realistic optical microscopy simulations in biological tissues

PubMed Central

Glaser, Adam K.; Chen, Ye; Liu, Jonathan T.C.

2017-01-01

Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method’s flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy. PMID:28983499

Optical spectroscopy and microscopy of radiation-induced light-emitting point defects in lithium fluoride crystals and films

NASA Astrophysics Data System (ADS)

Montereali, R. M.; Bonfigli, F.; Menchini, F.; Vincenti, M. A.

2012-08-01

Broad-band light-emitting radiation-induced F2 and F3+ electronic point defects, which are stable and laser-active at room temperature in lithium fluoride crystals and films, are used in dosimeters, tuneable color-center lasers, broad-band miniaturized light sources and novel radiation imaging detectors. A brief review of their photoemission properties is presented, and their behavior at liquid nitrogen temperatures is discussed. Some experimental data from optical spectroscopy and fluorescence microscopy of these radiation-induced point defects in LiF crystals and thin films are used to obtain information about the coloration curves, the efficiency of point defect formation, the effects of photo-bleaching processes, etc. Control of the local formation, stabilization, and transformation of radiation-induced light-emitting defect centers is crucial for the development of optically active micro-components and nanostructures. Some of the advantages of low temperature measurements for novel confocal laser scanning fluorescence microscopy techniques, widely used for spatial mapping of these point defects through the optical reading of their visible photoluminescence, are highlighted.

Induced structural defects in Ti-doped ZnO and its two-photon-excitation

NASA Astrophysics Data System (ADS)

Martínez Julca, Milton A.; Rivera, Ivonnemary; Santillan Mercado, Jaime; Sierra, Heidy; Perales-Pérez, Oscar

2016-03-01

ZnO is a well-known luminescent material that reacts with light to generate free radicals enabling its use in cancer treatment by Photodynamic Therapy (PDT). Unfortunately, up to know, the photo-excitation of ZnO-based materials' requires excitation with ultraviolet light, which limits their biomedical applications. In this regard, this work investigates the effect of Ti species incorporation into the lattice of ZnO nanoparticles (NPs) with the aim of improving the corresponding optical properties and enabling the two-photoexcitation with 690nm-light (near infrared light). A modified polyol-based route was used to synthesize pure and Ti-doped (9% at.) ZnO NPs. X-ray diffraction confirmed the formation of ZnO-wurtzite whereas Scanning Electron Microscopy confirmed the formation of monodispersed 100-nm NPs. Raman Spectroscopy measurements evidenced the presence of zinc interstitials (Zni) and oxygen vacancies (VO) in the host oxide strcuture. Asynthesized NPs were excited using the technique of two-photon fluorescence microscopy (TPFM). The photoluminescence (PL) spectra generated from the analysis of TPFM images revealed a high emission peak presence in the green region (555 nm) that was assigned to VO. Also, a weak but noticeable band at 420 nm was detected, which is attributed to electron transition from the shallow donor level of Zni to the valence band. These PL transitions will favor triplet states formation necessary to yield cytotoxic reactive oxygen species. Furthermore, the presence of the PL peaks confirmed the Ti-ZnO NPs capacity to be excited by 690-nm light, thus, opening new possibilities for this NPs to be used in lightinduced bio-medical applications.

Properties of new diffusion filters for treatment of amblyopia with accurate occlusive effects.

PubMed

Sasaki, Makoto; Iwasaki, Tsuneto; Kondo, Hiroyuki; Tawara, Akihiko

2016-06-01

Our purpose is to present the characteristics of newly developed diffusion filters that can reduce the best-corrected visual acuity (BCVA) of the non-amblyopic eye to a specified value and that can be used to treat amblyopia. Silica sol is a colorless and transparent colloidal gel of different particle sizes. The silica was added to an emulsion adhesive, thoroughly mixed, and coated evenly on polyethylene terephthalate films. Twelve filters with 12 different concentrations of silica were constructed. The density of the silica particles on the films was determined by scanning electron microscopy, and the haze values and light transmittance were measured with a goniophotometer. The reduction of the BCVA by the filters was determined in 16 healthy young women (mean age, 22.0 ± 2.3 years) by attaching the filters to spectacles. Scanning electron microscopy showed a monolayer of evenly spaced silica particles. The haze values of the 12 filters were related to the concentration of silica. The total light transmittance of the 12 filters was not significantly correlated to the concentration of silica. The BCVAs measured with the 12 filters were significantly and inversely correlated with the concentration of silica for both eyes (right eye, y = 0.174x - 0.197, R(2) = 0.951; left eye, y = 0.173x - 0.212, R(2) = 0.983). These findings indicate that these diffusion filters can reduce the BCVA with no reduction of light transmittance. We conclude that they can be used to degrade the image of the dominant eye by known amounts in patients with amblyopia without affecting the overall light levels to the eye, i.e., form deprivation without light deprivation.

Low-level laser therapy on MCF-7 cells: a micro-Fourier transform infrared spectroscopy study.

PubMed

Magrini, Taciana D; dos Santos, Nathalia Villa; Milazzotto, Marcella Pecora; Cerchiaro, Giselle; da Silva Martinho, Herculano

2012-10-01

Low-level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably scale from photobiostimulation/photobioinhibition at the cellular level to the molecular level. The detailed mechanism underlying this effect remains unknown. This study quantifies some relevant aspects of LLLT related to molecular and cellular variations. Malignant breast cells (MCF-7) were exposed to spatially filtered light from a He-Ne laser (633 nm) with fluences of 5, 28.8, and 1000  mJ/cm². The cell viability was evaluated by optical microscopy using the Trypan Blue viability test. The micro-Fourier transform infrared technique was employed to obtain the vibrational spectra of each experimental group (control and irradiated) and identify the relevant biochemical alterations that occurred due to the process. It was observed that the red light influenced the RNA, phosphate, and serine/threonine/tyrosine bands. We found that light can influence cell metabolism depending on the laser fluence. For 5  mJ/cm², MCF-7 cells suffer bioinhibition with decreased metabolic rates. In contrast, for the 1  J/cm² laser fluence, cells present biostimulation accompanied by a metabolic rate elevation. Surprisingly, at the intermediate fluence, 28.8  mJ/cm², the metabolic rate is increased despite the absence of proliferative results. The data were interpreted within the retrograde signaling pathway mechanism activated with light irradiation.

Light Microscopy at Maximal Precision

NASA Astrophysics Data System (ADS)

Bierbaum, Matthew; Leahy, Brian D.; Alemi, Alexander A.; Cohen, Itai; Sethna, James P.

2017-10-01

Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

NASA Astrophysics Data System (ADS)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Photonic modulation of EGFR: 280nm low level light arrests cancer cell activation and migration

NASA Astrophysics Data System (ADS)

Botelho, Cláudia M.; Marques, Rogério; Viruthachalam, Thiagarajan; Gonçalves, Odete; Vorum, Henrik; Gomes, Andreia C.; Neves-Petersen, Maria Teresa

2017-02-01

Overexpression of the Epidermal Growth Factor Receptor (EGFR) by cancer cells is associated with a poor prognosis for the patient. For several decades, therapies targeting EGFR have been designed, including the use of monoclonal antibodies and small molecule tyrosine kinase inhibitors. The use of these molecules had good clinical results, although its efficiency (and specificity) is still far from being optimal. In this paper, we present a new approach for a possible new cancer therapy targeting EGFR and using low intensity 280nm light. The influence of 280nm UVB illumination on cancer cells stimulated with 2nM of EGF was followed by time-lapse confocal microscopy. The 280nm illumination of the cancer cells blocks EGFR activation, inhibiting EGFR internalization and cell migration thus inhibiting the transition to the metastatic phenotype. Exposure time is a very important factor. The higher the illumination time the more significant differences were observed: 280nm light delayed or completely halted EGFR activation in the cell membrane, mainly at the cell junction level, and delayed or halted EGFR endocytic internalization, filopodia formation and cell migration.

Structural Investigation of Biological and Semiconductor Nanostructures with Nonlinear Multicontrast Microscopy

NASA Astrophysics Data System (ADS)

Cisek, Richard

Physical and functional properties of advanced nano-composite materials and biological structures are determined by self-organized atoms and molecules into nanostructures and in turn by microscopic organization of the nanostructures into assemblies of higher structural complexity. Therefore, microscopes are indispensable tools for structural investigations at various levels of organization. In this work, novel nonlinear optical microscopy methods were developed to non-invasively study structural organization at the nanoscopic and microscopic levels. Atomic organization of semiconductor nanowires, molecular organization of amylose biocrystallites in starch granules, and microscopic organization of several photosynthetic organisms was elucidated. The structure of ZnSe nanowires, key components in many modern nanodevices, was investigated using polarization harmonic generation microscopy. Based on nonlinear optical properties of the different crystal lattices, zinc blende and wurtzite nanowires were differentiated, and the three-dimensional orientation of the zinc blende nanowires could be found. The structure of starch granules, a model biocrystal, important in food as well as health sciences, was also investigated using polarization harmonic microscopy. The study was combined with ab initio calculations using the crystal structures of amylose A and B, revealing that second harmonic signals originate from the hydroxide and hydrogen bonds in the starch granules. Visualization of several photosynthetic organisms including the green algae, Chlamydomonas reinhardtii, two species of cyanobacteria, Leptolyngbya sp. and Anabaena sp., aggregates of light-harvesting pigment-protein complexes as well as chloroplasts from green plants were also explored, revealing that future nonlinear microscopy applications could include structural studies of cell walls, the Chlamydomonas eyespot, and photosynthetic membranes. In this study, several nonlinear optical microscopy modalities were developed for quantitative structural investigations of nano and micro-sized architectures. Non-invasive extraction of crystallographic information in microscopic samples will have a number of potential benefits, for example, in clinical applications, allowing observations of disease states inside tissues without the need for biopsy. Industrial nanotechnology will benefit from fast determination of nanostructures with nonlinear microscopy that will improve quality of nanodevices.

High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

PubMed

Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

2015-12-01

In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Extremely strong self-assembly of a bimetallic salen complex visualized at the single-molecule level.

PubMed

Salassa, Giovanni; Coenen, Michiel J J; Wezenberg, Sander J; Hendriksen, Bas L M; Speller, Sylvia; Elemans, Johannes A A W; Kleij, Arjan W

2012-04-25

A bis-Zn(salphen) structure shows extremely strong self-assembly both in solution as well as at the solid-liquid interface as evidenced by scanning tunneling microscopy, competitive UV-vis and fluorescence titrations, dynamic light scattering, and transmission electron microscopy. Density functional theory analysis on the Zn(2) complex rationalizes the very high stability of the self-assembled structures provoked by unusual oligomeric (Zn-O)(n) coordination motifs within the assembly. This coordination mode is strikingly different when compared with mononuclear Zn(salphen) analogues that form dimeric structures having a typical Zn(2)O(2) central unit. The high stability of the multinuclear structure therefore holds great promise for the development of stable self-assembled monolayers with potential for new opto-electronic materials.

Apoptosis of rat kidney cells after 241-americium administration.

PubMed

Labéjof, L; Berry, J P; Duchambon, P; Poncy, J L; Galle, P

1998-01-01

Tumors induction by americium is well known but there are no data on the biological effects of this radionucleide at subcellular level. In order to study the possible ultrastructural lesions induced by this element, a group of rats were injected with 241-Americium-citrate (9 kBq), once a week for five weeks and sacrificed 7 days after the last injection. We describe the alterations observed in the cortex kidney using cytochemical (TUNEL reaction) and histochemical (PAS staining) methods for light microscopy as well as electron microscopy techniques. Various types of lesions were detected: condensation of nuclear chromatine, fragmentation of the nuclei, swollen mitochondria, disappearance of mitochondrial crests and skrinking of the cytoplasm. This study clearly demonstrated the induction of apoptosis by americium in rat cortex kidney cells.

Identification of oleoresin in epoxy-embedded slash pine tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Birchem, R.; Brown, C.L.

1978-01-01

Sudan black B stains oleoresin blue-black in epoxy-embedded material as well as in living tissue. The Sudan black B staining properties of oleoresin are similar to those of lipid, but it can be distinguished from tannin, which stains brown. Practically all oleoresin present in resin ducts and intercellular spaces, and much of that contained in epithelial and ray cells, is extracted in preparatory procedures for electron microscopy. A fixation procedure is proposed which preserves significantly more oleoresin in situ. The use of Sudan black B enables one to localize oleoresin by light microscopy, and permits direct comparison of adjacent sectionsmore » of epoxy-embedded material at the ultrastructure level. Ultrastructurally oleoresin and lipid possess similar electron densities and can be distinguished from the highly electron-opaque tannin deposits.« less

Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

PubMed

de Carvalho, Marcelo Pires Nogueira; Queiroz-Hazarbassanov, Nicolle Gilda Teixeira; de Oliveira Massoco, Cristina; Sant'Anna, Sávio Stefanini; Lourenço, Mariana Mathias; Levin, Gabriel; Sogayar, Mari Cleide; Grego, Kathleen Fernandes; Catão-Dias, José Luiz

2017-09-01

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tattoo ink nanoparticles in skin tissue and fibroblasts

PubMed Central

Twigg, Peter C; Baker, Richard; Tobin, Desmond J

2015-01-01

Summary Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells. PMID:26171294

Nanocrystals of [Cu3(btc)2] (HKUST-1): a combined time-resolved light scattering and scanning electron microscopy study.

PubMed

Zacher, Denise; Liu, Jianing; Huber, Klaus; Fischer, Roland A

2009-03-07

The formation of [Cu(3)(btc)(2)] (HKUST-1; btc = 1,3,5-benzenetricarboxylate) nanocrystals from a super-saturated mother solution at room temperature was monitored by time-resolved light scattering (TLS); the system is characterized by a rapid growth up to a size limit of 200 nm within a few minutes, and the size and shape of the crystallites were also determined by scanning electron microscopy (SEM).

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes[C][W

PubMed Central

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I.

2013-01-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation. PMID:23898030

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

PubMed

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I

2013-06-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

Curcumin Inhibits Tau Aggregation and Disintegrates Preformed Tau Filaments in vitro.

PubMed

Rane, Jitendra Subhash; Bhaumik, Prasenjit; Panda, Dulal

2017-01-01

The pathological aggregation of tau is a common feature of most of the neuronal disorders including frontotemporal dementia, Parkinson's disease, and Alzheimer's disease. The inhibition of tau aggregation is considered to be one of the important strategies for treating these neurodegenerative diseases. Curcumin, a natural polyphenolic molecule, has been reported to have neuroprotective ability. In this work, curcumin was found to bind to adult tau and fetal tau with a dissociation constant of 3.3±0.4 and 8±1 μM, respectively. Molecular docking studies indicated a putative binding site of curcumin in the microtubule-binding region of tau. Using several complementary techniques, including dynamic light scattering, thioflavin S fluorescence, 90° light scattering, electron microscopy, and atomic force microscopy, curcumin was found to inhibit the aggregation of tau. The dynamic light scattering analysis and atomic force microscopic images revealed that curcumin inhibits the oligomerization of tau. Curcumin also disintegrated preformed tau oligomers. Using Far-UV circular dichroism, curcumin was found to inhibit the β-sheets formation in tau indicating that curcumin inhibits an initial step of tau aggregation. In addition, curcumin inhibited tau fibril formation. Furthermore, the effect of curcumin on the preformed tau filaments was analyzed by atomic force microscopy, transmission electron microscopy, and 90° light scattering. Curcumin treatment disintegrated preformed tau filaments. The results indicated that curcumin inhibited the oligomerization of tau and could disaggregate tau filaments.

The use of light-emitting diode fluorescence to diagnose mycobacterial lymphadenitis in fine-needle aspirates from children

PubMed Central

van Wyk, A. C.; Marais, B. J.; Warren, R. M.; van Wyk, S. S.; Wright, C. A.

2011-01-01

SUMMARY BACKGROUND Fine-needle aspiration biopsy (FNAB) is a simple, safe and effective method for investigating suspected mycobacterial lymphadenitis in children. Fluorescence microscopy can provide rapid mycobacterial confirmation. Light-emitting diodes (LEDs) provide a cheap and robust excitation light source, making fluorescence microscopy feasible in resource-limited settings. OBJECTIVE To compare the diagnostic performance of LED fluorescence microscopy on Papanicolaou (PAP) stained smears with the conventional mercury vapour lamp (MVL). METHODS FNAB smears routinely collected from palpable lymph nodes in children with suspected mycobacterial disease were PAP-stained and evaluated by two independent microscopists using different excitatory light sources (MVL and LED). Mycobacterial culture results provided the reference standard. A manually rechargeable battery-powered LED power source was evaluated in a random subset. RESULTS We evaluated 182 FNAB smears from 121 children (median age 31 months, interquartile range 10–67). Mycobacterial cultures were positive in 84 of 121 (69%) children. The mean sensitivity with LED (mains-powered), LED (rechargeable battery-powered) and MVL was respectively 48.2%, 50.0% and 51.8% (specificity 78.4%, 86.7% and 78.4%). Inter-observer variation was similar for LED and MVL (κ = 0.5). CONCLUSION LED fluorescence microscopy provides a reliable alternative to conventional methods and has many favourable attributes that would facilitate improved, decentralised diagnostic services. PMID:21276297

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

PubMed

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

Super-resolution photoacoustic microscopy using a localized near-field of a plasmonic nanoaperture: a three-dimensional simulation study

NASA Astrophysics Data System (ADS)

Park, Byullee; Lee, Hongki; Upputuri, Paul Kumar; Pramanik, Manojit; Kim, Donghyun; Kim, Chulhong

2018-02-01

Super-resolution microscopy has been increasingly important to delineate nanoscale biological structures or nanoparticles. With these increasing demands, several imaging modalities, including super-resolution fluorescence microscope (SRFM) and electron microscope (EM), have been developed and commercialized. These modalities achieve nanoscale resolution, however, SRFM cannot image without fluorescence, and sample preparation of EM is not suitable for biological specimens. To overcome those disadvantages, we have numerically studied the possibility of superresolution photoacoustic microscopy (SR-PAM) based on near-field localization of light. Photoacoustic (PA) signal is generally acquired based on optical absorption contrast; thus it requires no agents or pre-processing for the samples. The lateral resolution of the conventional photoacoustic microscopy is limited to 200 nm by diffraction limit, therefore reducing the lateral resolution is a major research impetus. Our approach to breaking resolution limit is to use laser pulses of extremely small spot size as a light source. In this research, we simulated the PA signal by constructing the three dimensional SR-PAM system environment using the k-Wave toolbox. As the light source, we simulated ultrashort light pulses using geometrical nanoaperture with near-field localization of surface plasmons. Through the PA simulation, we have successfully distinguish cuboids spaced 3 nm apart. In the near future, we will develop the SR-PAM and it will contribute to biomedical and material sciences.

Cerebral vessels segmentation for light-sheet microscopy image using convolutional neural networks

NASA Astrophysics Data System (ADS)

Hu, Chaoen; Hui, Hui; Wang, Shuo; Dong, Di; Liu, Xia; Yang, Xin; Tian, Jie

2017-03-01

Cerebral vessel segmentation is an important step in image analysis for brain function and brain disease studies. To extract all the cerebrovascular patterns, including arteries and capillaries, some filter-based methods are used to segment vessels. However, the design of accurate and robust vessel segmentation algorithms is still challenging, due to the variety and complexity of images, especially in cerebral blood vessel segmentation. In this work, we addressed a problem of automatic and robust segmentation of cerebral micro-vessels structures in cerebrovascular images acquired by light-sheet microscope for mouse. To segment micro-vessels in large-scale image data, we proposed a convolutional neural networks (CNNs) architecture trained by 1.58 million pixels with manual label. Three convolutional layers and one fully connected layer were used in the CNNs model. We extracted a patch of size 32x32 pixels in each acquired brain vessel image as training data set to feed into CNNs for classification. This network was trained to output the probability that the center pixel of input patch belongs to vessel structures. To build the CNNs architecture, a series of mouse brain vascular images acquired from a commercial light sheet fluorescence microscopy (LSFM) system were used for training the model. The experimental results demonstrated that our approach is a promising method for effectively segmenting micro-vessels structures in cerebrovascular images with vessel-dense, nonuniform gray-level and long-scale contrast regions.

Microscopic video observation of capillary vessel systems using diffuse back lighting

NASA Astrophysics Data System (ADS)

Sakai, Minako; Arai, Hiroki; Iwai, Toshiaki

2017-04-01

We have been developing a simple and practical video microscopy system based on absorption spectra of biological substance to perform spectroscopic observation of living tissues. The diffuse backlighting effect is actively used in the developed system, which is generated by multiple light scattering in the tissue. It is demonstrated that the light specularly reflected from the skin surface can be completely suppressed in the microscopic observation and the biological activity of the capillary vessel systems distributed under the skin can be successfully observed. As a result, we can confirm the effectiveness of the video microscopy system using diffuse backlighting and the applicability of our developed system.

Analysis of off-axis incoherent digital holographic microscopy

NASA Astrophysics Data System (ADS)

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

Off-axis incoherent digital holography that enables single-shot three-dimensional (3D) distribution is introduced in the paper. Conventional fluorescence microscopy images 3D fields by sectioning, this prevents instant imaging of fast reactions of living cells. In order to realize digital holography from incoherent light, we adapted common path configuration to achieve the best temporal coherence. And by introducing gratings, we shifted the direction of each light to achieve off-axis interference. Simulations and preliminary experiments using LED light have confirmed the results. We expect to use this method to realize 3D phase imaging and fluorescent imaging at the same time from the same biological sample.

Thermal Images of Seeds Obtained at Different Depths by Photoacoustic Microscopy (PAM)

NASA Astrophysics Data System (ADS)

Domínguez-Pacheco, A.; Hernández-Aguilar, C.; Cruz-Orea, A.

2015-06-01

The objective of the present study was to obtain thermal images of a broccoli seed ( Brassica oleracea) by photoacoustic microscopy, at different modulation frequencies of the incident light beam ((0.5, 1, 5, and 20) Hz). The thermal images obtained in the amplitude of the photoacoustic signal vary with each applied frequency. In the lowest light frequency modulation, there is greater thermal wave penetration in the sample. Likewise, the photoacoustic signal is modified according to the structural characteristics of the sample and the modulation frequency of the incident light. Different structural components could be seen by photothermal techniques, as shown in the present study.

High-resolution corneal topography and tomography of fish eye using wide-field white light interference microscopy

NASA Astrophysics Data System (ADS)

Srivastava, Vishal; Nandy, Sreyankar; Singh Mehta, Dalip

2013-04-01

Topography and tomography of fish cornea is reconstructed using high resolution white light interference microscopy. White light interferograms at different depths were recorded by moving the object axially. For each depth position, five phase shifted interferograms were recorded and analyzed. From the reconstructed phase maps, the corneal topography and hence the refractive index was determined and from amplitude images the cross-sectional image of fish cornea was reconstructed. In the present method, we utilize a nearly common-path interference microscope and wide field illumination and hence do not require any mechanical B-scan. Therefore, the phase stability of the recorded data is improved.

Integrated light and scanning electron microscopy of GFP-expressing cells.

PubMed

Peddie, Christopher J; Liv, Nalan; Hoogenboom, Jacob P; Collinson, Lucy M

2014-01-01

Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes. © 2014 Elsevier Inc. All rights reserved.

Studying the mechanism of neurostimulation by infrared laser light using GCaMP6s and Rhodamine B imaging

NASA Astrophysics Data System (ADS)

Moreau, David; Lefort, Claire; Bardet, Sylvia M.; O'Connor, Rodney P.

2016-03-01

Infrared laser light radiation can be used to depolarize neurons and to stimulate neural activity. The absorption of infrared radiation and heating of biological tissue is thought to be the underlying mechanism of this phenomenon whereby local temperature increases in the plasma membrane of cells either directly influence membrane properties or act via temperature sensitive ion channels. Action potentials are typically measured electrically in neurons with microelectrodes, but they can also be observed using fluorescence microscopy techniques that use synthetic or genetically encoded calcium indicators. In this work, we studied the impact of infrared laser light on neuronal calcium signals to address the mechanism of these thermal effects. Cultured primary mouse hippocampal neurons expressing the genetically encoded calcium indicator GCaMP6s were used in combination with the temperature sensitive fluorophore Rhodamine B to measure calcium signals and temperature changes at the cellular level. Here we present our all-optical strategy for studying the influence of infrared laser light on neuronal activity.

Circumventing photodamage in live-cell microscopy

PubMed Central

Magidson, Valentin; Khodjakov, Alexey

2013-01-01

Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

Wave optics theory and 3-D deconvolution for the light field microscope

PubMed Central

Broxton, Michael; Grosenick, Logan; Yang, Samuel; Cohen, Noy; Andalman, Aaron; Deisseroth, Karl; Levoy, Marc

2013-01-01

Light field microscopy is a new technique for high-speed volumetric imaging of weakly scattering or fluorescent specimens. It employs an array of microlenses to trade off spatial resolution against angular resolution, thereby allowing a 4-D light field to be captured using a single photographic exposure without the need for scanning. The recorded light field can then be used to computationally reconstruct a full volume. In this paper, we present an optical model for light field microscopy based on wave optics, instead of previously reported ray optics models. We also present a 3-D deconvolution method for light field microscopy that is able to reconstruct volumes at higher spatial resolution, and with better optical sectioning, than previously reported. To accomplish this, we take advantage of the dense spatio-angular sampling provided by a microlens array at axial positions away from the native object plane. This dense sampling permits us to decode aliasing present in the light field to reconstruct high-frequency information. We formulate our method as an inverse problem for reconstructing the 3-D volume, which we solve using a GPU-accelerated iterative algorithm. Theoretical limits on the depth-dependent lateral resolution of the reconstructed volumes are derived. We show that these limits are in good agreement with experimental results on a standard USAF 1951 resolution target. Finally, we present 3-D reconstructions of pollen grains that demonstrate the improvements in fidelity made possible by our method. PMID:24150383

Evaluation of agave fiber delignification by means of microscopy techniques and image analysis.

PubMed

Hernández-Hernández, Hilda M; Chanona-Pérez, Jorge J; Calderón-Domínguez, Georgina; Perea-Flores, María J; Mendoza-Pérez, Jorge A; Vega, Alberto; Ligero, Pablo; Palacios-González, Eduardo; Farrera-Rebollo, Reynold R

2014-10-01

Recently, the use of different types of natural fibers to produce paper and textiles from agave plants has been proposed. Agave atrovirens can be a good source of cellulose and lignin; nevertheless, the microstructural changes that happen during delignification have scarcely been studied. The aim of this work was to study the microstructural changes that occur during the delignification of agave fibers by means of microscopy techniques and image analysis. The fibers of A. atrovirens were obtained from leaves using convective drying, milling, and sieving. Fibers were processed using the Acetosolv pulping method at different concentrations of acetic acid; increasing acid concentration promoted higher levels of delignification, structural damage, and the breakdown of fiber clumps. Delignification followed by spectrometric analysis and microstructural studies were carried out by light, confocal laser scanning and scanning electron microscopy and showed that the delignification process follows three stages: initial, bulk, and residual. Microscopy techniques and image analysis were efficient tools for microstructural characterization during delignification of agave fibers, allowing quantitative evaluation of the process and the development of linear prediction models. The data obtained integrated numerical and microstructural information that could be valuable for the study of pulping of lignocellulosic materials.

Low-energy shock wave preconditioning reduces renal ischemic reperfusion injury caused by renal artery occlusion.

PubMed

Xue, Yuquan; Xu, Zhibin; Chen, Haiwen; Gan, Weimin; Chong, Tie

2017-07-01

To evaluate whether low energy shock wave preconditioning could reduce renal ischemic reperfusion injury caused by renal artery occlusion. The right kidneys of 64 male Sprague Dawley rats were removed to establish an isolated kidney model. The rats were then divided into four treatment groups: Group 1 was the sham treatment group; Group 2, received only low-energy (12 kv, 1 Hz, 200 times) shock wave preconditioning; Group 3 received the same low-energy shock wave preconditioning as Group 2, and then the left renal artery was occluded for 45 minutes; and Group 4 had the left renal artery occluded for 45 minutes. At 24 hours and one-week time points after reperfusion, serum inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), creatinine (Cr), and cystatin C (Cys C) levels were measured, malondialdehyde (MDA) in kidney tissue was detected, and changes in nephric morphology were evaluated by light and electron microscopy. Twenty-four hours after reperfusion, serum iNOS, NGAL, Cr, Cys C, and MDA levels in Group 3 were significantly lower than those in Group 4; light and electron microscopy showed that the renal tissue injury in Group 3 was significantly lighter than that in Group 4. One week after reperfusion, serum NGAL, KIM-1, and Cys C levels in Group 3 were significantly lower than those in Group 4. Low-energy shock wave preconditioning can reduce renal ischemic reperfusion injury caused by renal artery occlusion in an isolated kidney rat model.

Image analysis applied to luminescence microscopy

NASA Astrophysics Data System (ADS)

Maire, Eric; Lelievre-Berna, Eddy; Fafeur, Veronique; Vandenbunder, Bernard

1998-04-01

We have developed a novel approach to study luminescent light emission during migration of living cells by low-light imaging techniques. The equipment consists in an anti-vibration table with a hole for a direct output under the frame of an inverted microscope. The image is directly captured by an ultra low- light level photon-counting camera equipped with an image intensifier coupled by an optical fiber to a CCD sensor. This installation is dedicated to measure in a dynamic manner the effect of SF/HGF (Scatter Factor/Hepatocyte Growth Factor) both on activation of gene promoter elements and on cell motility. Epithelial cells were stably transfected with promoter elements containing Ets transcription factor-binding sites driving a luciferase reporter gene. Luminescent light emitted by individual cells was measured by image analysis. Images of luminescent spots were acquired with a high aperture objective and time exposure of 10 - 30 min in photon-counting mode. The sensitivity of the camera was adjusted to a high value which required the use of a segmentation algorithm dedicated to eliminate the background noise. Hence, image segmentation and treatments by mathematical morphology were particularly indicated in these experimental conditions. In order to estimate the orientation of cells during their migration, we used a dedicated skeleton algorithm applied to the oblong spots of variable intensities emitted by the cells. Kinetic changes of luminescent sources, distance and speed of migration were recorded and then correlated with cellular morphological changes for each spot. Our results highlight the usefulness of the mathematical morphology to quantify kinetic changes in luminescence microscopy.

Biomolecule-controlled hydrothermal synthesis of C-N-S-tridoped TiO2 nanocrystalline photocatalysts for NO removal under simulated solar light irradiation.

PubMed

Wang, Yawen; Huang, Yu; Ho, Wingkei; Zhang, Lizhi; Zou, Zhigang; Lee, Shuncheng

2009-09-30

In this study, C-N-S-tridoped titanium dioxide (TiO(2)) nanocrystals were synthesized by using a facile hydrothermal method in the presence of a biomolecule l-cysteine. This biomolecule could not only serve as the common source for the carbon, sulfur and nitrogen tridoping, but also could control the final crystal phases and morphology. The resulting materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), nitrogen adsorption and UV-vis diffuse reflectance spectroscopy. XPS analysis revealed that S was incorporated into the lattice of TiO(2) through substituting oxygen atoms, N might coexist in the forms of N-Ti-O and Ti-O-N in tridoped TiO(2) and most C could form a mixed layer of carbonate species deposited on the surface of TiO(2) nanoparticles. The photocatalytic activities of the samples were tested on the removal of NO at typical indoor air level in a flow system under simulated solar light irradiation. The tridoped TiO(2) samples showed much higher removal efficiency than commercial P25 and the undoped counterpart photocatalyst. The enhanced visible light photocatalytic activity of C-N-S-tridoped TiO(2) nanocrystals was explained on the basis of characterizations. The possible formation process of the monodispersed C-N-S-tridoped anatase TiO(2) nanocrystals was also proposed. This study provides a new method to prepare visible light active TiO(2) photocatalyst.

Developing methods based on light sheet fluorescence microscopy for biophysical investigations of larval zebrafish

NASA Astrophysics Data System (ADS)

Taormina, Michael J.

Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges. A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish). The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo.

A facile hydrothermal approach to synthesize rGO/BiVO4 photocatalysts for visible light induced degradation of RhB dye

NASA Astrophysics Data System (ADS)

Pal, Shreyasi; Dutta, Shibsankar; De, Sukanta

2018-05-01

RGO/BiVO4 composites were synthesized by a simple hydrothermal method. The samples were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM) and surface analysis (BET). The photocatalytic activity of the as-prepared samples was evaluated by studying the degradation of model dyes rhodamine B (RhB) under visible light. The prepared rGO/BiVO4 composites exhibited higher photocatalytic activity for the degradation of RhB with a maximum removal rate of 86% under visible light irradiation under visible-light irradiation than pure BiVO4 nanoparticles (63%). This behavior could be associated to their higher specific surface area (BET), increased light absorption intensity and the degradation of electron-hole pair recombination in BiVO4 with the introduction of the rGO.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

PubMed Central

Bertrand, Vincent; Lenne, Pierre-François

2014-01-01

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time. PMID:24836407

Biomechanical and Histopathologic Effects of Pulsed-Light Accelerated Epithelium-On/-Off Corneal Collagen Cross-Linking.

PubMed

Zhang, Xiaoyu; Sun, Ling; Shen, Yang; Tian, Mi; Zhao, Jing; Zhao, Yu; Li, Meiyan; Zhou, Xingtao

2017-07-01

This study aimed to compare the biomechanical and histopathologic effects of transepithelial and accelerated epithelium-off pulsed-light accelerated corneal collagen cross-linking (CXL). A total of 24 New Zealand rabbits were analyzed after sham operation (control) or transepithelial or epithelium-off operation (45 mW/cm for both). The transepithelial group was treated with pulsed-light ultraviolet A for 5 minutes 20 seconds, and the epithelium-off group was treated for 90 seconds. Biomechanical testing, including ultimate stress, Young modulus, and the physiological modulus, was analyzed. Histological changes were evaluated by light microscopy and transmission electron microscopy. The stress-strain curve was nonlinear in both accelerated transepithelial and epithelium-off CXL groups. The stress and elastic moduli were all significantly higher in both experimental groups compared with the control group (P < 0.05), whereas there were no significant differences between the 2 treatment groups (P > 0.05). Six months after the operation, hematoxylin and eosin staining and transmission electron microscopy showed that the subcutaneous collagen fibers were arranged in a regular pattern, and the fiber density was higher in the experimental groups. Both transepithelial and accelerated epithelium-off CXL produced biomechanical and histopathologic improvements, which were not significantly different between the 2 pulsed-light accelerated CXL treatments.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

PubMed Central

Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

2016-01-01

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM. PMID:27977021

Nonvolatile RRAM cells from polymeric composites embedding recycled SiC powders.

PubMed

De Girolamo Del Mauro, Anna; Nenna, Giuseppe; Miscioscia, Riccardo; Freda, Cesare; Portofino, Sabrina; Galvagno, Sergio; Minarini, Carla

2014-10-21

Silicon carbide powders have been synthesized from tires utilizing a patented recycling process. Dynamic light scattering, Raman spectroscopy, SEM microscopy, and X-ray diffraction have been carried out to gather knowledge about powders and the final composite structure. The obtained powder has been proven to induce resistive switching in a PMMA polymer-based composite device. Memory effect has been detected in two-terminal devices having coplanar contacts and quantified by read-write-erase measurements in terms of level separation and persistence.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Woo Taek; Franceschi, V.R.; Okita, T.W.

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.

Polarized Light Microscopy

NASA Technical Reports Server (NTRS)

Frandsen, Athela F.

2016-01-01

Polarized light microscopy (PLM) is a technique which employs the use of polarizing filters to obtain substantial optical property information about the material which is being observed. This information can be combined with other microscopy techniques to confirm or elucidate the identity of an unknown material, determine whether a particular contaminant is present (as with asbestos analysis), or to provide important information that can be used to refine a manufacturing or chemical process. PLM was the major microscopy technique in use for identification of materials for nearly a century since its introduction in 1834 by William Fox Talbot, as other techniques such as SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared spectroscopy), XPD (X-ray Powder Diffraction), and TEM (Transmission Electron Microscopy) had not yet been developed. Today, it is still the only technique approved by the Environmental Protection Agency (EPA) for asbestos analysis, and is often the technique first applied for identification of unknown materials. PLM uses different configurations in order to determine different material properties. With each configuration additional clues can be gathered, leading to a conclusion of material identity. With no polarizing filter, the microscope can be used just as a stereo optical microscope, and view qualities such as morphology, size, and number of phases. With a single polarizing filter (single polars), additional properties can be established, such as pleochroism, individual refractive indices, and dispersion staining. With two polarizing filters (crossed polars), even more can be deduced: isotropy vs. anisotropy, extinction angle, birefringence/degree of birefringence, sign of elongation, and anomalous polarization colors, among others. With the use of PLM many of these properties can be determined in a matter of seconds, even for those who are not highly trained. McCrone, a leader in the field of polarized light microscopy, often advised, If you cant determine a specific optical property of a particle after two minutes, move onto another configuration. Since optical properties can be seen so very quickly and easily under polarized light, it is only necessary to spend a maximum of two minutes on a technique to determine a particular property, though often only a few seconds are required.

Immunophenotype of leukemic blasts with small peroxidase-positive granules detected by electron microscopy.

PubMed

Vainchenker, W; Villeval, J L; Tabilio, A; Matamis, H; Karianakis, G; Guichard, J; Henri, A; Vernant, J P; Rochant, H; Breton-Gorius, J

1988-05-01

Forty-three cases of undifferentiated leukemias by light microscopy examination were diagnosed as acute myeloblastic leukemias by ultrastructural revelation of peroxidase and were subsequently studied by immunological markers. In 41 of these cases, blasts were labeled by at least one of the antimyeloid MoAbs (My 7, My 9, and 80H5). An antimyeloperoxidase polyclonal antibody was used in 23 cases and was clearly positive in 11 of them, while cytochemistry by light microscopy was negative. These myeloblasts were frequently mixed with a minority of blasts from other lineages especially promegakaryoblasts. It is noteworthy that in 6 cases myeloid and lymphoid markers (E rosette receptor, common acute lymphoblastic leukemia antigen (cALLA), CD 9, CD 19 antigens (anti-B4 MoAb] were detected on a fraction of blast cells, suggesting a bilineage leukemia. However, in double labeling experiments, blasts with myeloperoxidase coexpressed lymphoid and myeloid markers including cALLA and CD 19 antigen. In one case, blasts had a typical non-B, non-T acute lymphoblastic leukemia phenotype (HLA-DR, CD 9, CD 19, cALLA positive) without staining by any of the antimyeloid MoAbs. However, 70% of the blasts were labeled by the antimyeloperoxidase antibody and expressed peroxidase-positive granules at ultrastructural level. In conclusion, most of the AML undiagnosed by optical cytochemistry are identified by antimyeloid antibodies. Some of these cases are also stained by some antilymphoid MoAbs. Use of antibodies against myeloperoxidase may improve the diagnosis of difficult cases of acute myeloblastic leukemia.

Early development of fern gametophytes in microgravity

NASA Technical Reports Server (NTRS)

Roux, Stanley J.; Chatterjee, Ani; Hillier, Sheila; Cannon, Tom

2003-01-01

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth. c2002 Published by Elsevier Science Ltd on behalf of COSPAR.

Microstructure and nutrient distribution in oats: influence on quality

NASA Astrophysics Data System (ADS)

Miller, S. Shea; Frégeau-Reid, Judith

2009-05-01

Oats have long been recognized as having superior quality among cereals with respect to protein and lipid composition as well as soluble dietary fibre (β-glucan). The microstructure and chemistry of oats influence oat quality, and thus are determinants of the end products derived from oats. Light and scanning electron microscopies have been used to elucidate microstructure and nutrient distribution in oats. The influence of variation in these parameters on oat quality can be demonstrated, from milling through to oat products for consumption. Milling quality is determined in part by hull architecture. SEM examination of oat hulls can help predict ease of dehulling, which affects the efficiency and economics of oat milling. In addition to protein and lipid, β-glucan is an important nutritional component of oats. Fluorescence microscopy can reveal both the relative amount and distribution of β-glucan in oat kernels. Consumption of oats or oat products containing β-glucan has been shown to have beneficial effects on carbohydrate and lipid metabolism. These health benefits have generated a demand for new and palatable ways to incorporate oats into the diet as consumer demand increases. To help meet this need, we have been investigating the use of micronized naked oats as a whole grain to be cooked and consumed as a rice alternative. Different varieties of naked oats had dramatically different acceptance levels from a sensory panel. SEM of the pericarp, light microscopy of the endosperm, and analyses of starch properties of the different varieties revealed differences that corresponded with sensory data.

Electronic, optical and photocatalytic behavior of Mn, N doped and co-doped TiO{sub 2}: Experiment and simulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ya Fei; Li, Can, E-mail: canli1983@gmail.com; Lu, Song

2016-03-15

The crystal phase structure, surface morphology, chemical states and optical properties of Mn, N mono-doped and co-doped TiO{sub 2} nanoparticles were investigated by X-ray powder diffractometry, Raman spectra, scanning electron microscopy, X-ray photoelectron spectroscopy and UV–vis diffuse reflectance spectroscopy. Meanwhile, geometry structures, formation energies, electronic and optical properties of all systems have been also analyzed by density functional theory. The results showed that the band gap values and the carrier mobility in the valence band, conduction band and impurity levels have a synergetic influence on the visible-light absorption and photocatalytic activity of the doped TiO{sub 2}. The number and themore » carrier mobility of impurity level jointly influence the photocatalytic activity of catalyst under visible-light. Especially, the photocatalytic activity of Mn-2N co-doped TiO{sub 2} beyond three-fold than that of pure TiO{sub 2} under visible-light. - Graphical abstract: The ILs formed by N-2p orbital in N single doped specimen lie above the VB, while the ILs formed by Mn-3d orbital in Mn single doped specimen appear below the CB. However, a large amount of ILs formed by N-2p orbital and Mn-3d orbital in N and Mn codoped specimens. The band gap values and the carrier mobility in the valence band, conduction band and impurity levels have a synergetic influence on the visible-light absorption and photocatalytic activity of the doped TiO{sub 2}. The number and the carrier mobility of impurity level jointly influence the photocatalytic activity of catalyst under visible-light.« less

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.

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

NASA Astrophysics Data System (ADS)

Cerbino, Roberto; Cicuta, Pietro

2017-09-01

Differential dynamic microscopy (DDM) is a technique that exploits optical microscopy to obtain local, multi-scale quantitative information about dynamic samples, in most cases without user intervention. It is proving extremely useful in understanding dynamics in liquid suspensions, soft materials, cells, and tissues. In DDM, image sequences are analyzed via a combination of image differences and spatial Fourier transforms to obtain information equivalent to that obtained by means of light scattering techniques. Compared to light scattering, DDM offers obvious advantages, principally (a) simplicity of the setup; (b) possibility of removing static contributions along the optical path; (c) power of simultaneous different microscopy contrast mechanisms; and (d) flexibility of choosing an analysis region, analogous to a scattering volume. For many questions, DDM has also advantages compared to segmentation/tracking approaches and to correlation techniques like particle image velocimetry. The very straightforward DDM approach, originally demonstrated with bright field microscopy of aqueous colloids, has lately been used to probe a variety of other complex fluids and biological systems with many different imaging methods, including dark-field, differential interference contrast, wide-field, light-sheet, and confocal microscopy. The number of adopting groups is rapidly increasing and so are the applications. Here, we briefly recall the working principles of DDM, we highlight its advantages and limitations, we outline recent experimental breakthroughs, and we provide a perspective on future challenges and directions. DDM can become a standard primary tool in every laboratory equipped with a microscope, at the very least as a first bias-free automated evaluation of the dynamics in a system.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Bending the Rules: Widefield Microscopy and the Abbe Limit of Resolution

PubMed Central

Verdaasdonk, Jolien S.; Stephens, Andrew D.; Haase, Julian; Bloom, Kerry

2014-01-01

One of the most fundamental concepts of microscopy is that of resolution–the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope–deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy. PMID:23893718

Performance of rapid diagnostic test, blood-film microscopy and PCR for the diagnosis of malaria infection among febrile children from Korogwe District, Tanzania.

PubMed

Mahende, Coline; Ngasala, Billy; Lusingu, John; Yong, Tai-Soon; Lushino, Paminus; Lemnge, Martha; Mmbando, Bruno; Premji, Zul

2016-07-26

Rapid diagnostic tests (RDT) and light microscopy are still recommended for diagnosis to guide the clinical management of malaria despite difficult challenges in rural settings. The performance of these tests may be affected by several factors, including malaria prevalence and intensity of transmission. The study evaluated the diagnostic performance of malaria RDT, light microscopy and polymerase chain reaction (PCR) in detecting malaria infections among febrile children at outpatient clinic in Korogwe District, northeastern Tanzania. The study enrolled children aged 2-59 months with fever and/or history of fever in the previous 48 h attending outpatient clinics. Blood samples were collected for identification of Plasmodium falciparum infection using histidine-rich-protein-2 (HRP-2)-based malaria RDT, light microscopy and conventional PCR. A total of 867 febrile patients were enrolled into the study. Malaria-positive samples were 85/867 (9.8 %, 95 % CI, 7.9-12.0 %) by RDT, 72/867 (8.3 %, 95 % CI, 6.5-10.1 %) by microscopy and 79/677 (11.7 %, 95 % CI, 9.3-14.3 %) by PCR. The performance of malaria RDT compared with microscopy results had sensitivity and positive predictive value (PPV) of 88.9 % (95 % CI, 79.3-95.1 %) and 75.3 % (95 % CI, 64.8-84.0 %), respectively. Confirmation of P. falciparum infection with PCR analysis provided lower sensitivity and PPV of 88.6 % (95 % CI, 79.5-94.7 %) and 84.3 % (95 % CI, 74.7-91.4 %) for RDT compared to microscopy. Diagnosis of malaria infection is still a challenge due to variation in results among diagnostic methods. HRP-2 malaria RDT and microscopy were less sensitive than PCR. Diagnostic tools with high sensitivity are required in areas of low malaria transmission.

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Label-free volumetric optical imaging of intact murine brains

NASA Astrophysics Data System (ADS)

Ren, Jian; Choi, Heejin; Chung, Kwanghun; Bouma, Brett E.

2017-04-01

A central effort of today’s neuroscience is to study the brain’s ’wiring diagram’. The nervous system is believed to be a network of neurons interacting with each other through synaptic connection between axons and dendrites, therefore the neuronal connectivity map not only depicts the underlying anatomy, but also has important behavioral implications. Different approaches have been utilized to decipher neuronal circuits, including electron microscopy (EM) and light microscopy (LM). However, these approaches typically demand extensive sectioning and reconstruction for a brain sample. Recently, tissue clearing methods have enabled the investigation of a fully assembled biological system with greatly improved light penetration. Yet, most of these implementations, still require either genetic or exogenous contrast labeling for light microscopy. Here we demonstrate a high-speed approach, termed as Clearing Assisted Scattering Tomography (CAST), where intact brains can be imaged at optical resolution without labeling by leveraging tissue clearing and the scattering contrast of optical frequency domain imaging (OFDI).

In vivo imaging of cardiac development and function in zebrafish using light sheet microscopy.

PubMed

Weber, Michael; Huisken, Jan

2015-01-01

Detailed studies of heart development and function are crucial for our understanding of cardiac failures and pave the way for better diagnostics and treatment. However, the constant motion and close incorporation into the cardiovascular system prevent in vivo studies of the living, unperturbed heart. The complementary strengths of the zebrafish model and light sheet microscopy provide a useful platform to fill this gap. High-resolution images of the embryonic vertebrate heart are now recorded from within the living animal: deep inside the unperturbed heart we can follow cardiac contractions and measure action potentials and calcium transients. Three-dimensional reconstructions of the entire beating heart with cellular resolution give new insights into its ever-changing morphology and facilitate studies into how individual cells form the complex cardiac network. In addition, cardiac dynamics and robustness are now examined with targeted optical manipulation. Overall, the combination of zebrafish and light sheet microscopy represents a promising addition for cardiac research and opens the door to a better understanding of heart function and development.

Ultrastructural pathogenesis of lesions produced by exposure to oxygen difluoride with correlative light microscopy

NASA Technical Reports Server (NTRS)

Harrison, G.; Mackenzie, W.

1973-01-01

The lungs of rats exposed to OF2 were examined by light and electron microscopy. The exposures were for 30 to 60 minutes to an average of 4.5 ppm OF2, the minimal lethal dose. Animals were sacrificed after 30 (group 1) and 60 minutes (group 2) exposure and 1 (group 3) and 2 (group 4) hours following 60 minutes exposure. Mild gross changes were observed in groups 3 and 4, but no light microscopic lesions were found. Alterations were noted in all four groups using electron microscopy. These were mostly indicative of fluid change and consisted of blebbing of the endothelial and epithelial layers of the alveolocapillary wall and rarification of the cytoplasm of these cells. The lamellar bodies of the Type II cells showed an increasing and consistent loss of matrix structure and density. These fine structural changes increased in quantity and severity as time of exposure or post-exposure period increased. (Modified author abstract)

High-resolution light-sheet microscopy: a simulation of an optical illumination system for oil immersion

NASA Astrophysics Data System (ADS)

Lu, Xiang; Heintzmann, Rainer; Leischner, Ulrich

2015-09-01

Light sheet microscopy is a microscopy technique characterized by an illumination from the side, perpendicular to the direction of observation. While this is often used and easy to implement for imaging samples with water-immersion, the application in combination with oil-immersion is less often used and requires a specific optimization. In this paper we present our design of a light-sheet illumination optical system with a ~1μm illumination thickness, a long working distance through the immersion oil, and including a focusing system allowing for moving the focus-spot of the lightsheet laterally through the field of view. This optical design allows for the acquisition of fluorescence images in 3D with isotropic resolution of below 1 micrometer of whole-mount samples with a size of ~1mm diameter. This technique enables high-resolution insights in the 3D structure of biological samples, e.g. for research of insect anatomy or for imaging of biopsies in medical diagnostics.

Onset of molar incisor hypomineralization (MIH).

PubMed

Fagrell, Tobias G; Salmon, Phil; Melin, Lisa; Norén, Jörgen G

2013-01-01

The etiological factors and timing of the onset of molar incisor hypomineralization (MIH) are still not clear. The aim of this study was to examine ground radial and sagittal sections from teeth diagnosed with MIH using light microscopy, polarized light microscopy and X-ray micro-computed tomography (XMCT) and to estimate the onset and timing of the MIH and to relate the hypomineralized enamel to the incremental lines. Thirteen extracted permanent first molars diagnosed MIH, were analyzed with light microscopy and XMCT. The hypomineralized areas were mainly located in the mesio-buccal cusps, starting at the enamel-dentin-junction and continuing towards the enamel surface. In a relative gray scale analysis the values decreased from the EDJ towards the enamel surface. The findings indicate that the ameloblasts in the hypomineralized enamel are capable of forming an enamel of normal thickness, but with a substantial reduction of their capacity for maturation of enamel. Chronologically, it is estimated that the timing of the disturbance is at a period during the first 6-7 months of age.

Light Sheet Fluorescence Microscopy (LSFM)

PubMed Central

Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A.J.

2015-01-01

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. PMID:25559221

Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy

NASA Astrophysics Data System (ADS)

Lawrence, Keelan; Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

2018-02-01

Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.

Ultrafast Imaging of Chiral Surface Plasmon by Photoemission Electron Microscopy

NASA Astrophysics Data System (ADS)

Dai, Yanan; Dabrowski, Maciej; Petek, Hrvoje

We employ Time-Resolved Photoemission Electron Microscopy (TR-PEEM) to study surface plasmon polariton (SPP) wave packet dynamics launched by tunable (VIS-UV) femtosecond pulses of various linear and circular polarizations. The plasmonic structures are micron size single-crystalline Ag islands grown in situ on Si surfaces and characterized by Low Energy Electron Microscopy (LEEM). The local fields of plasmonic modes enhance two and three photon photoemission (2PP and 3PP) at the regions of strong field enhancement. Imaging of the photoemission signal with PEEM electron optics thus images the plasmonic fields excited in the samples. The observed PEEM images with left and right circularly polarized light show chiral images, which is a consequence of the transverse spin momentum of surface plasmon. By changing incident light polarization, the plasmon interference pattern shifts with light ellipticity indicating a polarization dependent excitation phase of SPP. In addition, interferometric-time resolved measurements record the asymmetric SPP wave packet motion in order to characterize the dynamical properties of chiral SPP wave packets.

The analysis of colored acrylic, cotton, and wool textile fibers using micro-Raman spectroscopy. Part 2: comparison with the traditional methods of fiber examination.

PubMed

Buzzini, Patrick; Massonnet, Genevieve

2015-05-01

In the second part of this survey, the ability of micro-Raman spectroscopy to discriminate 180 fiber samples of blue, black, and red cottons, wools, and acrylics was compared to that gathered with the traditional methods for the examination of textile fibers in a forensic context (including light microscopy methods, UV-vis microspectrophotometry and thin-layer chromatography). This study shows that the Raman technique plays a complementary and useful role to obtain further discriminations after the application of light microscopy methods and UV-vis microspectrophotometry and assure the nondestructive nature of the analytical sequence. These additional discriminations were observed despite the lower discriminating powers of Raman data considered individually, compared to those of light microscopy and UV-vis MSP. This study also confirms that an instrument equipped with several laser lines is necessary for an efficient use as applied to the examination of textile fibers in a forensic setting. © 2015 American Academy of Forensic Sciences.

Exploring the brain on multiple scales with correlative two-photon and light sheet microscopy

NASA Astrophysics Data System (ADS)

Silvestri, Ludovico; Allegra Mascaro, Anna Letizia; Costantini, Irene; Sacconi, Leonardo; Pavone, Francesco S.

2014-02-01

One of the unique features of the brain is that its activity cannot be framed in a single spatio-temporal scale, but rather spans many orders of magnitude both in space and time. A single imaging technique can reveal only a small part of this complex machinery. To obtain a more comprehensive view of brain functionality, complementary approaches should be combined into a correlative framework. Here, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, taking advantage of blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living thy1-GFP-M mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from the apical portion, the whole pyramidal neuron can then be segmented. The correlative approach presented here allows contextualizing within a three-dimensional anatomic framework the neurons whose dynamics have been observed with high detail in vivo.

Study on validity of a rapid diagnostic test kit versus light microscopy for malaria diagnosis in Ahmedabad city, India.

PubMed

Vyas, S; Puwar, B; Patel, V; Bhatt, G; Kulkarni, S; Fancy, M

2014-05-01

Light microscopy of blood smears for diagnosis of malaria in the field has several limitations, notably delays in diagnosis. This study in Ahmedabad in Gujarat State, India, evaluated the diagnostic performance of a rapid diagnostic test for malaria (SD Bioline Malaria Ag P.f/Pan) versus blood smear examination as the gold standard. All fever cases presenting at 13 urban health centres were subjected to rapid diagnostic testing and thick and thin blood smears. A total of 677 cases with fever were examined; 135 (20.0%) tested positive by rapid diagnostic test and 86 (12.7%) by blood smear. The sensitivity of the rapid diagnostic test for malaria was 98.8%, specificity was 91.5%, positive predictive value 63.0% and negative predictive value 99.8%. For detection of Plasmodium falciparum the sensitivity of rapid diagnostic test was 100% and specificity was 97.3%. The results show the acceptability of the rapid test as an alternative to light microscopy in the field setting.

Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images

PubMed Central

Salas, Desirée; Le Gall, Antoine; Fiche, Jean-Bernard; Valeri, Alessandro; Ke, Yonggang; Bron, Patrick; Bellot, Gaetan

2017-01-01

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions. PMID:28811371

Topography and refractometry of nanostructures using spatial light interference microscopy.

PubMed

Wang, Zhuo; Chun, Ik Su; Li, Xiuling; Ong, Zhun-Yong; Pop, Eric; Millet, Larry; Gillette, Martha; Popescu, Gabriel

2010-01-15

Spatial light interference microscopy (SLIM) is a novel method developed in our laboratory that provides quantitative phase images of transparent structures with a 0.3 nm spatial and 0.03 nm temporal accuracy owing to the white light illumination and its common path interferometric geometry. We exploit these features and demonstrate SLIM's ability to perform topography at a single atomic layer in graphene. Further, using a decoupling procedure that we developed for cylindrical structures, we extract the axially averaged refractive index of semiconductor nanotubes and a neurite of a live hippocampal neuron in culture. We believe that this study will set the basis for novel high-throughput topography and refractometry of man-made and biological nanostructures.

Phase contrast scanning transmission electron microscopy imaging of light and heavy atoms at the limit of contrast and resolution.

PubMed

Yücelen, Emrah; Lazić, Ivan; Bosch, Eric G T

2018-02-08

Using state of the art scanning transmission electron microscopy (STEM) it is nowadays possible to directly image single atomic columns at sub-Å resolution. In standard (high angle) annular dark field STEM ((HA)ADF-STEM), however, light elements are usually invisible when imaged together with heavier elements in one image. Here we demonstrate the capability of the recently introduced Integrated Differential Phase Contrast STEM (iDPC-STEM) technique to image both light and heavy atoms in a thin sample at sub-Å resolution. We use the technique to resolve both the Gallium and Nitrogen dumbbells in a GaN crystal in [[Formula: see text

Correlative Light and Scanning X-Ray Scattering Microscopy of Healthy and Pathologic Human Bone Sections

PubMed Central

Giannini, C.; Siliqi, D.; Bunk, O.; Beraudi, A.; Ladisa, M.; Altamura, D.; Stea, S.; Baruffaldi, F.

2012-01-01

Scanning small and wide angle X-ray scattering (scanning SWAXS) experiments were performed on healthy and pathologic human bone sections. Via crystallographic tools the data were transformed into quantitative images and as such compared with circularly polarized light (CPL) microscopy images. SWAXS and CPL images allowed extracting information of the mineral nanocrystalline phase embedded, with and without preferred orientation, in the collagen fibrils, mapping local changes at sub-osteon resolution. This favorable combination has been applied for the first time to biopsies of dwarfism syndrome and Paget's disease to shed light onto the cortical structure of natural bone in healthy and pathologic sections. PMID:22666538

Use of astronomy filters in fluorescence microscopy.

PubMed

Piper, Jörg

2012-02-01

Monochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this "low energy excitation," photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes.

PubMed

Wüstner, Daniel; Brewer, Jonathan R; Bagatolli, Luis; Sage, Daniel

2011-01-01

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols. © 2010 Wiley-Liss, Inc.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Differential localization of SAP102 and PSD-95 is revealed in hippocampal spines using super-resolution light microscopy.

PubMed

Zheng, Chan-Ying; Wang, Ya-Xia; Kachar, Bechara; Petralia, Ronald S

2011-01-01

Synapse-associated protein 102 (SAP102) and postsynaptic density 95 (PSD-95) are two major cytoskeleton proteins in the postsynaptic density (PSD). Both of them belong to the membrane-associated guanylate kinase (MAGUK) family, which clusters and anchors glutamate receptors and other proteins at synapses. In our previous study, we found that SAP102 and PSD-95 have different distributions, using combined light/electron microscopy (LM/EM) methods.1 Here, we double labeled endogenous SAP102 and PSD-95 in mature hippocampal neurons, and then took images by two different kinds of super resolution microscopy-Stimulated Emission Depletion microscopy (STED) and DeltaVision OMX 3D super resolution microscopy. We found that our 2D and 3D super resolution data were consistent with our previous LM/EM data, showing significant differences in the localization of SAP102 and PSD-95 in spines: SAP102 is distributed in both the PSD and cytoplasm of spines, while PSD-95 is concentrated only in the PSD area. These results indicate functional differences between SAP102 and PSD-95 in synaptic organization and plasticity.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Multiphoton microscopy and image guided light activated therapy using nanomaterials (Conference Presentation)

NASA Astrophysics Data System (ADS)

Prasad, Paras N.

2017-02-01

This talk will focus on design and applications of nanomaterials exhibiting strong multiphoton upconversion for multiphoton microscopy as well as for image-guided and light activated therapy .1-3 Such processes can occur by truly nonlinear optical interactions proceeding through virtual intermediate states or by stepwise coupled linear excitations through real intermediate states. Multiphoton processes in biocompatible multifunctional nanoparticles allow for 3D deep tissue imaging. In addition, they can produce in-situ photon conversion of deep tissue penetrating near IR light into a needed shorter wavelength light for photo-activated therapy at a targeted site, thus overcoming the limited penetration of UV or visible light into biological media. We are using near IR emitters such as silicon quantum dots which also exhibit strong multiphoton excitation for multiphoton microscopy. Another approach involves nonlinear nanocrystals such as ZnO which can produce four wave mixing, sum frequency generation as well as second harmonic generation to convert a deep tissue penetrating Near IR light at the targeted biological site to a desired shorter wavelength light suitable for bio imaging or activation of a therapy. We have utilized this approach to activate a photosensitizer for photodynamic therapy. Yet another type of upconversion materials is rare-earth ion doped optical nanotransformers which transform a Near IR (NIR) light from an external source by sequential single photon absorption, in situ and on demand, to a needed wavelength. Applications of these nanotransformers in multiphoton photoacoustic imaging will also be presented. An exciting direction pursued by us using these multiphoton nanoparticles, is functional imaging of brain. Simultaneously, they can effect optogenetics for regioselective stimulation of neurons for providing an effective intervention/augmentation strategy to enhance the cognitive state and lead to a foundation for futuristic vision of super human capabilities. Challenges and opportunities will be discussed.

An easy-to-implement filter for separating photo-excited signals from topography in scanning tunneling microscopy.

PubMed

Wang, Kangkang; Rosenmann, Daniel; Holt, Martin; Winarski, Robert; Hla, Saw-Wai; Rose, Volker

2013-06-01

In order to achieve elemental and chemical sensitivity in scanning tunneling microscopy (STM), synchrotron x-rays have been applied to excite core-level electrons during tunneling. The x-ray photo-excitations result in tip currents that are superimposed onto conventional tunneling currents. While carrying important physical information, the varying x-ray induced currents can destabilize the feedback loop causing it to be unable to maintain a constant tunneling current, sometimes even causing the tip to retract fully or crash. In this paper, we report on an easy-to-implement filter circuit that can separate the x-ray induced currents from conventional tunneling currents, thereby allowing simultaneous measurements of topography and chemical contrasts. The filter and the schematic presented here can also be applied to other variants of light-assisted STM such as laser STM.

A line scanned light-sheet microscope with phase shaped self-reconstructing beams.

PubMed

Fahrbach, Florian O; Rohrbach, Alexander

2010-11-08

We recently demonstrated that Microscopy with Self-Reconstructing Beams (MISERB) increases both image quality and penetration depth of illumination beams in strongly scattering media. Based on the concept of line scanned light-sheet microscopy, we present an add-on module to a standard inverted microscope using a scanned beam that is shaped in phase and amplitude by a spatial light modulator. We explain technical details of the setup as well as of the holograms for the creation, positioning and scaling of static light-sheets, Gaussian beams and Bessel beams. The comparison of images from identical sample areas illuminated by different beams allows a precise assessment of the interconnection between beam shape and image quality. The superior propagation ability of Bessel beams through inhomogeneous media is demonstrated by measurements on various scattering media.

Photodynamic action of methylene blue in osteosarcoma cells in vitro.

PubMed

Guan, Jiemin; Lai, Xiaoping; Wang, Xinna; Leung, Albert Wingnang; Zhang, Hongwei; Xu, Chuanshan

2014-03-01

Osteosarcoma is a common malignant bone tumor which threatens the life of young people worldwide. To explore alternative strategy for combating osteosarcoma, a light-emitting diode (LED) that activates methylene blue (MB) was used in the present study to investigate cell death of osteosarcoma-derived UMR106 cells. Photocytotoxicity in UMR106 cells was investigated 24h after photodynamic activation of MB using sulforhodamine B (SRB) assay and light microscopy. Apoptosis induction was observed 24h after photodynamic treatment using a confocal laser scanning microscopy (CLSM) with Hoechst 33342 staining. The change in mitochondrial membrane potential (MMP) was analyzed using a flow cytometry with rhodamine 123 staining. MB under red light irradiation caused a drug-concentration (0-100μM) and light-dose (0-32J/cm(2)) dependent cytotoxicity in UMR106 cells. The SRB assay and light microscopy observed a significant decrease in the number of UMR106 cells attached to the bottom of culture well after LED light-activated MB (100μM, 32J/cm(2)). Nuclear shrinkage, chromatin condensation and fragmentation were found in the treated cells by nuclear staining. In addition, flow cytometry showed that the MMP in UMR106 cells was rapidly reduced by photo-activated MB (100μM, 32J/cm(2)). Photodynamic action of MB under LED irradiation could remarkably kill osteosarcoma cells and induce cell apoptosis as well as MMP collapse. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

PubMed

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

Asymmetric Flow-Field Flow Fractionation (AF4) of Aqueous C60 Aggregates with Dynamic Light Scattering Size and LC-MS

EPA Science Inventory

Current methods for the size determination of nanomaterials in aqueous suspension include dynamic or static light scattering and electron or atomic force microscopy techniques. Light scattering techniques are limited by poor resolution and the scattering intensity dependence on p...

Potential concerns with analytical Methods Used for the detection of Batrachochytrium salamandrivorans from archived DNA of amphibian swab samples, Oregon, USA

USGS Publications Warehouse

Iwanowicz, Deborah; Olson, Deanna H.; Adams, Michael J.; Adams, Cynthia; Anderson, Chauncey; Blaustein, Andrew R; Densmore, Christine L.; Figiel, Chester; Schill, William B.; Chestnut, Tara

2017-01-01

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010–2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts.

Analysis of Long Bone and Vertebral Failure Patterns.

DTIC Science & Technology

1982-09-30

processes further supported the findings of • :the scanning electron microscopy studies . In the impacted animals, the cartilage surface was eroded... cartilage matrix. In the six years post-impaction group, the articular cartilage had converted to fibrocartilage instead of normal hyaline cartilage . The...columns of four rhesus monkeys have been collected and are being processed for study with light microscopy and scanning electron microscopy. The baboon

A simple method for maintaining relative positions of separate tissue elements during processing for electron microscopy.

PubMed

Stirling, C A

1978-09-01

Molten (328 K) 20% gelatin is used as a 'glue' to hold together separate tissue elements or tissue elements that may be separated when cutting small blocks of tissue for plastic embedding. Standard aldehyde and osmium fixation, dehydration and epoxy embedding are compatible with this as is semi-thin sectioning for light microscopy or thin sectioning for electron microscopy.

High Prevalence of Human Liver Infection by Amphimerus spp. Flukes, Ecuador

PubMed Central

Calvopiña, Manuel; Cevallos, William; Kumazawa, Hideo; Eisenberg, Joseph

2011-01-01

Amphimerus spp. flukes are known to infect mammals, but human infections have not been confirmed. Microscopy of fecal samples from 397 persons from Ecuador revealed Opisthorchiidae eggs in 71 (24%) persons. Light microscopy of adult worms and scanning electron microscopy of eggs were compatible with descriptions of Amphimerus spp. This pathogen was only observed in communities that consumed undercooked fish. PMID:22172165

A light-dependent ammonia-assimilating mechanism in the ctenidia of a giant clam

NASA Astrophysics Data System (ADS)

Hiong, Kum C.; Choo, Celine Y. L.; Boo, Mel V.; Ching, Biyun; Wong, Wai P.; Chew, Shit F.; Ip, Yuen K.

2017-03-01

Giant clams harbor symbiotic zooxanthellae and manifest light-enhanced calcification. In light, they also increase the absorption and assimilation of exogenous ammonia, but the roles of the host and symbionts are unclear and the reason for light dependency remains enigmatic. Here, we report the complete coding cDNA sequence of a glutamine synthetase (GS), from the ctenidia (gill) of Tridacna squamosa. The deduced GS amino acid sequence was closely related to other animal GSs, indicating a host origin. The GS/GS transcript level and protein abundance increased significantly in the ctenidia after 12 h of light exposure. These results denote the ctenidia as the site of light-enhanced ammonia absorption with the absorbed ammonia being assimilated to glutamine by the host GS. Immunofluorescence microscopy confirmed that GS was expressed predominantly in the cytoplasm of the epithelial cells separating the tertiary water channels from the hemolymph inside the ctenidia. Hence, unlike symbiotic cnidarians, the host clam probably supplies exogenous nitrogen as glutamine from the ctenidia, through the hemolymph and tubular fluid, to the nitrogen-deficient zooxanthellae which reside extracellularly in the extensible outer mantle. This is the first report on light-dependent gene and protein expression of a host enzyme involved in nitrogen metabolism in an alga-invertebrate association.

A new genus and species of Nematalycidae (Acari: Endeostigmata)

USDA-ARS?s Scientific Manuscript database

Osperalycus tenerphagus, a new genus and species of Nematalycidae (Acari: Endeostigmata), is described from Ohio, USA, using light microscopy and low temperature scanning electron microscopy. Specimens were extracted from two different loam soils. This genus can be readily distinguished from the oth...

Evaluation of airborne asbestos exposure from routine handling of asbestos-containing wire gauze pads in the research laboratory.

PubMed

Garcia, Ediberto; Newfang, Daniel; Coyle, Jayme P; Blake, Charles L; Spencer, John W; Burrelli, Leonard G; Johnson, Giffe T; Harbison, Raymond D

2018-07-01

Three independently conducted asbestos exposure evaluations were conducted using wire gauze pads similar to standard practice in the laboratory setting. All testing occurred in a controlled atmosphere inside an enclosed chamber simulating a laboratory setting. Separate teams consisting of a laboratory technician, or technician and assistant simulated common tasks involving wire gauze pads, including heating and direct wire gauze manipulation. Area and personal air samples were collected and evaluated for asbestos consistent with the National Institute of Occupational Safety Health method 7400 and 7402, and the Asbestos Hazard Emergency Response Act (AHERA) method. Bulk gauze pad samples were analyzed by Polarized Light Microscopy and Transmission Electron Microscopy to determine asbestos content. Among air samples, chrysotile asbestos was the only fiber found in the first and third experiments, and tremolite asbestos for the second experiment. None of the air samples contained asbestos in concentrations above the current permissible regulatory levels promulgated by OSHA. These findings indicate that the level of asbestos exposure when working with wire gauze pads in the laboratory setting is much lower than levels associated with asbestosis or asbestos-related lung cancer and mesothelioma. Copyright © 2018. Published by Elsevier Inc.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

PubMed

Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

2012-09-01

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

PubMed

Mitchell, V; Sigala, J; Ballot, C; Jumeau, F; Barbotin, A L; Duhamel, A; Rives, N; Rigot, J M; Escalier, D; Peers, M C

2015-03-01

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk. © 2014 Blackwell Verlag GmbH.

Multi-spectral digital holographic microscopy for enhanced quantitative phase imaging of living cells

NASA Astrophysics Data System (ADS)

Kemper, Björn; Kastl, Lena; Schnekenburger, Jürgen; Ketelhut, Steffi

2018-02-01

Main restrictions of using laser light in digital holographic microscopy (DHM) are coherence induced noise and parasitic reflections in the experimental setup which limit resolution and measurement accuracy. We explored, if coherence properties of partial coherent light sources can be generated synthetically utilizing spectrally tunable lasers. The concept of the method is demonstrated by label-free quantitative phase imaging of living pancreatic tumor cells and utilizing an experimental configuration including a commercial microscope and a laser source with a broad tunable spectral range of more than 200 nm.

Photoassisted Kelvin probe force microscopy at GaN surfaces: The role of polarity

NASA Astrophysics Data System (ADS)

Wei, J. D.; Li, S. F.; Atamuratov, A.; Wehmann, H.-H.; Waag, A.

2010-10-01

The behavior of GaN surfaces during photoassisted Kelvin probe force microscopy is demonstrated to be strongly dependant on surface polarity. The surface photovoltage of GaN surfaces illuminated with above-band gap light is analyzed as a function of time and light intensity. Distinct differences between Ga-polar and N-polar surfaces could be identified, attributed to photoinduced chemisorption of oxygen during illumination. These differences can be used for a contactless, nondestructive, and easy-performable analysis of the polarity of GaN surfaces.

Microscopic characterization of tension wood cell walls of Japanese beech (Fagus crenata) treated with ionic liquids.

PubMed

Kanbayashi, Toru; Miyafuji, Hisashi

2016-09-01

Tension wood that is an abnormal part formed in angiosperms has been barely used for wood industry. In this study, to utilize the tension wood effectively by means of liquefaction using ionic liquid, we performed morphological and topochemical determination of the changes in tension wood of Japanese beech (Fagus crenata) during ionic liquid treatment at the cellular level using light microscopy, scanning electron microscopy and confocal Raman microscopy. Ionic liquid treatment induced cell wall swelling in tension wood. Changes in the tissue morphology treated with ionic liquids were different between normal wood and tension wood, moreover the types of ionic liquids. The ionic liquid 1-ethyl-3-methylimidazolium chloride liquefied gelatinous layers rapidly, whereas 1-ethylpyridinium bromide liquefied slowly but delignified selectively. These novel insights into the deconstruction behavior of tension wood cell walls during ionic liquid treatment provide better understanding of the liquefaction mechanism. The obtained knowledge will contribute to development of an effective chemical processing of tension wood using ionic liquids and lead to efficient use of wood resources. Copyright © 2016 Elsevier Ltd. All rights reserved.

Advantages of intermediate X-ray energies in Zernike phase contrast X-ray microscopy.

PubMed

Wang, Zhili; Gao, Kun; Chen, Jian; Hong, Youli; Ge, Xin; Wang, Dajiang; Pan, Zhiyun; Zhu, Peiping; Yun, Wenbing; Jacobsen, Chris; Wu, Ziyu

2013-01-01

Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (<1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy. Until now, X-ray microscopes operating in the "water window" energy range between carbon and oxygen k-shell absorption edges have produced outstanding 3D images of cryo-preserved cells. The relatively low X-ray energy (<540 eV) of the water window imposes two important limitations: limited penetration (<10 μm) not suitable for imaging larger cells or tissues, and small depth of focus (DoF) for high resolution 3D imaging (e.g., ~1 μm DoF for 20 nm resolution). An X-ray microscope operating at intermediate energy around 2.5 keV using Zernike phase contrast can overcome the above limitations and reduces radiation dose to the specimen. Using a hydrated model cell with an average chemical composition reported in literature, we calculated the image contrast and the radiation dose for absorption and Zernike phase contrast, respectively. The results show that an X-ray microscope operating at ~2.5 keV using Zernike phase contrast offers substantial advantages in terms of specimen size, radiation dose and depth-of-focus. Copyright © 2012 Elsevier Inc. All rights reserved.

Increased numbers of Demodex in contact lens wearers.

PubMed

Jalbert, Isabelle; Rejab, Shazana

2015-06-01

The aim of this study was to determine if Demodex infestation is more frequent in contact lens wearers than in nonwearers. Secondary aims were to evaluate the effects of Demodex on the ocular surface (symptoms and signs) and to evaluate the ability of confocal laser scanning microscopy to detect and quantify the Demodex infestation compared with the conventional light microscopic technique. Forty Asian female participants (20 nonwearers, 20 lens wearers) with a mean (± SD) age of 27 (± 9) years were recruited. Ocular comfort scores (Ocular Surface Disease Index, Ocular Comfort Index, and Dry Eye Questionnaire), vital staining (corneal, conjunctival, and lid wiper), tear osmolarity, tear breakup time, and meibomian gland evaluation were evaluated. Demodex was detected using in vivo confocal microscopy and conventional light microscopy. The number of Demodex was higher in lens wearers than in nonwearers (7.6 [± 5.8] vs. 5.0 [± 3.1]; p = 0.02). Demodex was observed in a large majority (90%) of lens wearers and in 65% of nonwearers using confocal microscopy (p = 0.06). The detection rate was lower in both groups using conventional light microscopy (p = 0.003) where Demodex could only be confirmed in 70% and 60% of lens wearers and nonwearers, respectively. The number of Demodex tended to increase with age (ρ = 0.28, p = 0.08), but Demodex did not appear to affect ocular comfort or any clinical signs (p > 0.05). Contact lens wearers harbor Demodex as frequently as nonwearers and in higher numbers, which is best detected using in vivo confocal microscopy. The significance of these findings is uncertain because no associations were found with any symptoms and signs of dry eye disease.

Comparative Confocal and Histopathological Study of Corneal Changes in Multiple Myeloma.

PubMed

Micali, Antonio; Roszkowska, Anna M; Postorino, Elisa I; Rania, Laura; Aragona, Emanuela; Wylegala, Edward; Nowinska, Anna; Ieni, Antonio; Calimeri, Sebastiano; Pisani, Antonina; Aragona, Pasquale; Puzzolo, Domenico

2017-01-01

Corneal opacities rarely occur in multiple myeloma (MM). Our study correlates the findings of in vivo confocal microscopy (IVCM), a useful diagnostic tool, with histopathological features of corneal opacities appearing in a patient with MM. Case report. A 53-year-old man developed corneal opacities in both eyes, more pronounced in the left eye. After IVCM examination, he underwent penetrating keratoplasty in the left eye, and the button was processed for light and electron microscopy and immunohistochemistry. The diagnosis of MM was made, as confirmed by the elevation of IgGk light chains. IVCM demonstrated hyperreflective areas at the epithelial level, hyperreflective keratocytes of dendritic and lamellar morphology in whole stroma, and hyperreflective endothelial cells. Histopathological examination disclosed many vacuoles in the epithelial cell cytoplasm and a homogenous granular material in the Bowman layer. In stroma, keratocytes of different shape and size, with vesicles laden with an abnormal material, were evident. In Descemet membrane, the posterior nonbanded zone had a honeycomb appearance because of the presence of many roundish spaces among wide-spaced collagen fibers. Endothelial cells demonstrated vesicles filled with a material of uneven electron density. Immunohistochemical analysis showed strong positivity for IgGk light chains in keratocytes and among stromal lamellae. This is the first study describing a correspondence between IVCM features and histopathological alterations observed in corneal opacities in MM. The results of this study improve the current understanding of the pictures obtained by IVCM studies.

Bioorthogonal Chemical Imaging for Biomedicine

NASA Astrophysics Data System (ADS)

Min, Wei

2017-06-01

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because relatively bulky fluorescent labels could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, we have developed a bioorthogonal chemical imaging platform. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, multiplicity and biocompatibility for imaging small biomolecules in live systems including tissues and organisms. Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. This bioorthogonal chemical imaging platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, further chemical and spectroscopic strategies allow for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species, bringing small molecules under the illumination of modern light microscopy.

Visualisation of collagen fibrils in joint cartilage using STIM

NASA Astrophysics Data System (ADS)

Reinert, T.; Reibetanz, U.; Vogt, J.; Butz, T.; Werner, A.; Gründer, W.

2001-07-01

The scanning transmission ion microscopy (STIM) method was used to investigate the collagen network structure of the articular cartilage from a pig's knee in comparison with high resolution nuclear magnetic resonance imaging (microscopic NMR-tomography) and polarised light microscopy (PLM). Single collagen fibrils down to 200 nm in diameter were visualised. It was proved that the cartilage collagen network consists partly of zones of oriented fibrils as suggested by NMR measurements. Radially oriented fibrils were found in the zone near the calcified zone (hypertrophic zone) of both tibia and femur, and in the tibial radial zone. Tangentially oriented fibrils were found in the femoral and tibial superficial zone and in a second zone of the femoral cartilage. Polarisation light microscopy reveals broader zones of orientation than it was found with STIM.

Comparative Analysis Reveals Potential Utility of Digital Microscopy in the Evaluation of Peripheral Blood Smears With Some Barriers to Implementation.

PubMed

Gomez-Gelvez, Juan C; Kryvenko, Oleksandr N; Chabot-Richards, Devon S; Foucar, Kathryn; Inamdar, Kedar V; Karner, Kristin H

2015-07-01

Evaluation of the peripheral blood smear (PBS) is an essential diagnostic test in current medical practice. We aimed to evaluate the use of digital microscopy for the examination of PBS as an option to provide expert interpretation to remote sites and in "on-call" situations. We collected 100 Wright-Giemsa-stained PBS slides representing normal and abnormal findings seen at a community-based hospital. Four hematopathologists independently evaluated the cases using conventional light and digital microscopy. When comparing digital vs light microscopy, most of the cellular features evaluated showed at least a moderate degree of agreement in at least three of the reviewers. Discrepancies in final diagnosis were identified in a minority of the cases, most of which were attributed to the poorer resolution of digital microscopy at high magnification (×400). These results support the limited use of digital microscopy for evaluation and triage of peripheral blood smears as a practical option to obtain expert opinion in locations where experienced staff is not available on site. Our results indicate that while digital microscopy is well suited for basic triage of these blood smears, limitations in quality of imaging at higher magnification as well as large file size may limit its utility in certain settings and situations. Copyright© by the American Society for Clinical Pathology.

3D single-molecule super-resolution microscopy with a tilted light sheet.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-01-09

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Topography and refractometry of nanostructures using spatial light interference microscopy (SLIM)

PubMed Central

Wang, Zhuo; Chun, Ik Su; Li, Xiuling; Ong, Zhun-Yong; Pop, Eric; Millet, Larry; Gillette, Martha; Popescu, Gabriel

2010-01-01

Spatial Light Interference Microscopy (SLIM) is a novel method developed in our laboratory that provides quantitative phase images of transparent structures with 0.3 nm spatial and 0.03 nm temporal accuracy owing to the white light illumination and its common path interferometric geometry. We exploit these features and demonstrate SLIM's ability to perform topography at a single atomic layer in graphene. Further, using a decoupling procedure that we developed for cylindrical structures, we extract the axially-averaged refractive index of semiconductor nanotubes and a neurite of a live hippocampal neuron in culture. We believe that this study will set the basis for novel high-throughput topography and refractometry of man-made and biological nanostructures. PMID:20081970

Programmable LED-based integrating sphere light source for wide-field fluorescence microscopy.

PubMed

Rehman, Aziz Ul; Anwer, Ayad G; Goldys, Ewa M

2017-12-01

Wide-field fluorescence microscopy commonly uses a mercury lamp, which has limited spectral capabilities. We designed and built a programmable integrating sphere light (PISL) source which consists of nine LEDs, light-collecting optics, a commercially available integrating sphere and a baffle. The PISL source is tuneable in the range 365-490nm with a uniform spatial profile and a sufficient power at the objective to carry out spectral imaging. We retrofitted a standard fluorescence inverted microscope DM IRB (Leica) with a PISL source by mounting it together with a highly sensitive low- noise CMOS camera. The capabilities of the setup have been demonstrated by carrying out multispectral autofluorescence imaging of live BV2 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Magnetic resonance microscopy of prostate tissue: How basic science can inform clinical imaging development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bourne, Roger

2013-03-15

This commentary outlines how magnetic resonance imaging (MRI) microscopy studies of prostate tissue samples and whole organs have shed light on a number of clinical imaging mysteries and may enable more effective development of new clinical imaging methods.

Multimodal Kelvin Probe Force Microscopy Investigations of a Photovoltaic WSe2/MoS2 Type-II Interface.

PubMed

Almadori, Yann; Bendiab, Nedjma; Grévin, Benjamin

2018-01-10

Atomically thin transition-metal dichalcogenides (TMDC) have become a new platform for the development of next-generation optoelectronic and light-harvesting devices. Here, we report a Kelvin probe force microscopy (KPFM) investigation carried out on a type-II photovoltaic heterojunction based on WSe 2 monolayer flakes and a bilayer MoS 2 film stacked in vertical configuration on a Si/SiO 2 substrate. Band offset characterized by a significant interfacial dipole is pointed out at the WSe 2 /MoS 2 vertical junction. The photocarrier generation process and phototransport are studied by applying a differential technique allowing to map directly two-dimensional images of the surface photovoltage (SPV) over the vertical heterojunctions (vHJ) and in its immediate vicinity. Differential SPV reveals the impact of chemical defects on the photocarrier generation and that negative charges diffuse in the MoS 2 a few hundreds of nanometers away from the vHJ. The analysis of the SPV data confirms unambiguously that light absorption results in the generation of free charge carriers that do not remain coulomb-bound at the type-II interface. A truly quantitative determination of the electron-hole (e-h) quasi-Fermi levels splitting (i.e., the open-circuit voltage) is achieved by measuring the differential vacuum-level shift over the WSe 2 flakes and the MoS 2 layer. The dependence of the energy-level splitting as a function of the optical power reveals that Shockley-Read-Hall processes significantly contribute to the interlayer recombination dynamics. Finally, a newly developed time-resolved mode of the KPFM is applied to map the SPV decay time constants. The time-resolved SPV images reveal the dynamics of delayed recombination processes originating from photocarriers trapping at the SiO 2 /TMDC interfaces.

Rapid Morphological Brain Abnormalities during Acute Methamphetamine Intoxication in the Rat. An Experimental study using Light and Electron Microscopy

PubMed Central

Sharma, Hari S.; Kiyatkin, Eugene A.

2009-01-01

This study describes morphological abnormalities of brain cells during acute methamphetamine (METH) intoxication in the rat and demonstrates the role of hyperthermia, disruption of the blood-brain barrier (BBB) and edema in their development. Rats with chronically implanted brain, muscle and skin temperature probes and an intravenous (iv) catheter were exposed to METH (9 mg/kg) at standard (23°C) and warm (29°C) ambient temperatures, allowing for the observation of hyperthermia ranging from mild to pathological levels (38–42°C). When brain temperature peaked or reached a level suggestive of possible lethality (>41.5°C), rats were injected with Evans blue (EB), rapidly anesthetized, perfused, and their brains were taken for further analyses. Four brain areas (cortex, hippocampus, thalamus and hypothalamus) were analyzed for EB extravasation, water and electrolyte (Na+, K+, Cl−) contents, immunostained for albumin and glial fibrillary acidic protein, and examined for neuronal, glial and axonal alterations using standard light and electron microscopy. These examinations revealed profound abnormalities in neuronal, glial, and endothelial cells, which were stronger with METH administered at 29°C than 23°C and tightly correlated with brain and body hyperthermia. These changes had some structural specificity, but in each structure they tightly correlated with increases in EB levels, the numbers of albumin-positive cells, and water and ion contents, suggesting leakage of the BBB, acutely developing brain edema, and serious shifts in brain ion homeostasis as leading factors underlying brain abnormalities. While most of these acute structural and functional abnormalities appear to be reversible, they could trigger subsequent cellular alterations in the brain and accelerate neurodegeneration—the most dangerous complication of chronic amphetamine-like drug abuse. PMID:18773954

Differential dynamic microscopy of bidisperse colloidal suspensions.

PubMed

Safari, Mohammad S; Poling-Skutvik, Ryan; Vekilov, Peter G; Conrad, Jacinta C

2017-01-01

Research tasks in microgravity include monitoring the dynamics of constituents of varying size and mobility in processes such as aggregation, phase separation, or self-assembly. We use differential dynamic microscopy, a method readily implemented with equipment available on the International Space Station, to simultaneously resolve the dynamics of particles of radius 50 nm and 1 μm in bidisperse aqueous suspensions. Whereas traditional dynamic light scattering fails to detect a signal from the larger particles at low concentrations, differential dynamic microscopy exhibits enhanced sensitivity in these conditions by accessing smaller wavevectors where scattering from the large particles is stronger. Interference patterns due to scattering from the large particles induce non-monotonic decay of the amplitude of the dynamic correlation function with the wavevector. We show that the position of the resulting minimum contains information on the vertical position of the particles. Together with the simple instrumental requirements, the enhanced sensitivity of differential dynamic microscopy makes it an appealing alternative to dynamic light scattering to characterize samples with complex dynamics.

Learning a cost function for microscope image segmentation.

PubMed

Nilufar, Sharmin; Perkins, Theodore J

2014-01-01

Quantitative analysis of microscopy images is increasingly important in clinical researchers' efforts to unravel the cellular and molecular determinants of disease, and for pathological analysis of tissue samples. Yet, manual segmentation and measurement of cells or other features in images remains the norm in many fields. We report on a new system that aims for robust and accurate semi-automated analysis of microscope images. A user interactively outlines one or more examples of a target object in a training image. We then learn a cost function for detecting more objects of the same type, either in the same or different images. The cost function is incorporated into an active contour model, which can efficiently determine optimal boundaries by dynamic programming. We validate our approach and compare it to some standard alternatives on three different types of microscopic images: light microscopy of blood cells, light microscopy of muscle tissue sections, and electron microscopy cross-sections of axons and their myelin sheaths.

Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

PubMed Central

Ngo, John T.; Adams, Stephen R.; Deerinck, Thomas J.; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F.; Bertozzi, Carolyn R.; Ellisman, Mark H.; Tsien, Roger Y.

2016-01-01

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy.

PubMed

Pozzi, P; Wilding, D; Soloviev, O; Verstraete, H; Bliek, L; Vdovin, G; Verhaegen, M

2017-01-23

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

Value of light microscopy to diagnose urogenital gonorrhoea: a diagnostic test study in Indonesian clinic-based and outreach sexually transmitted infections services.

PubMed

Hananta, I Putu Yuda; van Dam, Alje P; Bruisten, Sylvia Maria; van der Loeff, Maarten Franciscus Schim; Soebono, Hardyanto; Christiaan de Vries, Henry John

2017-08-11

Gonorrhoea is a common sexually transmitted disease caused by Neisseria gonorrhoeae (Ng) infection. Light microscopy of urogenital smears is used as a simple tool to diagnose urogenital gonorrhoea in many resource-limited settings. We aimed to evaluate the accuracy of light microscopy to diagnose urogenital gonorrhoea as compared with a PCR-based test. In 2014, we examined 632 male urethral and 360 endocervical smears in clinic-based and outreach settings in Jakarta, Yogyakarta and Denpasar, Indonesia. Using the detection of Ng DNA by a validated PCR as reference test, we evaluated the accuracy of two light microscopic criteria to diagnose urogenital gonorrhoea in genital smears: (1) the presence of intracellular Gram-negative diplococci (IGND) and (2) ≥5 polymorphonuclear leucocytes (PMNL)/oil-immersion field (oif) in urethral or ≥20 PMNL/oif in endocervical smears. In male urethral smears, IGND testing had a sensitivity (95% CI), specificity (95% CI) and kappa±SE of 59.0% (50.1 to 67.4), 89.4% (86.3 to 91.9) and 0.49±0.04, respectively. For PMNL count, these were 59.0% (50.1 to 67.4), 83.7% (80.2 to 86.9) and 0.40±0.04, respectively. The accuracy of IGND in the clinic-based settings (72.0% (57.5 to 83.3), 95.2% (91.8 to 97.5) and 0.68±0.06, respectively) was better than in the outreach settings (51.2% (40.0 to 62.3), 83.4% (78.2 to 87.8) and 0.35±0.06, respectively). In endocervical smears, light microscopy performed poorly regardless of the setting or symptomatology, with kappas ranging from -0.09 to 0.24. Light microscopy using IGND and PMNL criteria can be an option with moderate accuracy to diagnose urethral gonorrhoea among males in a clinic-based setting. The poor accuracy in detecting endocervical infections indicates an urgent need to implement advanced methods, such as PCR. Further investigations are needed to identify the poor diagnostic outcome in outreach services. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Multimodal lightsheet, structured illumination and Airyscan superresolution microscopy of chloroplast size and its impact on light propagation

USDA-ARS?s Scientific Manuscript database

Altering chloroplast size changes the way light propagates through a leaf by altering light reflectance and transmission as well as absorption by chlorophyll. Thus changing chloroplast size can used to manipulate leaf optical properties to optimize photosynthetic efficiency with the ultimate goal of...

Sub-diffraction limit resolution in microscopy

NASA Technical Reports Server (NTRS)

Cheng, Ming (Inventor); Chen, Weinong (Inventor)

2007-01-01

A method and apparatus for visualizing sub-micron size particles employs a polarizing microscope wherein a focused beam of polarized light is projected onto a target, and a portion of the illuminating light is blocked from reaching the specimen, whereby to produce a shadow region, and projecting diffracted light from the target onto the shadow region.

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

PubMed

Ochoa, Gina H; Clark, Ying Mei; Matsumoto, Brian; Torres-Ruiz, Jose A; Robles, Laura J

2002-02-01

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.

Imaging bacterial spores by soft-x-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stead, A.D.; Ford, T.W.; Judge, J.

1997-04-01

Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores bymore » soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.« less

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Light sensitive polymer obtained by dispersion of azo-functionalized POSS nanoparticles

NASA Astrophysics Data System (ADS)

Miniewicz, A.; Tomkowicz, M.; Karpinski, P.; Sznitko, L.; Mossety-Leszczak, B.; Dutkiewicz, M.

2015-07-01

Hybrid inorganic-organic nanoparticles based on cubic siloxane cage (RSiO3/2)8, known as polyhedral oligosilsesquioxane (POSS), have been functionalized by eight groups of azo-benzene mesogens and dispersed in poly(methyl methacrylate) PMMA matrix. Presence of azo-benzene units adds an important light-driven functionality to the system due to their photoisomerization resulting in refractive index and/or absorption changes of the whole system. The polymer films containing various concentrations of azo-POSS nanoparticles show remarkable changes of surface morphology being either transparent (at low POSS concentration) or highly scattering (at high POSS concentration) for visible light. Surface structures were examined by optical microscopy as well as by atomic force microscopy (AFM). Results of photoinduced alignment are discussed in the framework of light-induced modification of the aliphatic chains containing azo-benzene photoisomerizing moieties and self-organization process.

STM-induced light emission enhanced by weakly coupled organic ad-layers

NASA Astrophysics Data System (ADS)

Cottin, M. C.; Ekici, E.; Bobisch, C. A.

2018-03-01

We analyze the light emission induced by the tunneling current flowing in a scanning tunneling microscopy experiment. In particular, we study the influence of organic ad-layers on the light emission on the initial monolayer of bismuth (Bi) on Cu(111) in comparison to the well-known case of organic ad-layers on Ag(111). On the Bi/Cu(111)-surface, we find that the scanning tunneling microscopy-induced light emission is considerably enhanced if an organic layer, e.g., the fullerene C60 or the perylene derivate perylene-tetracarboxylic-dianhydride, is introduced into the tip-sample junction. The enhancement can be correlated with a peculiarly weak interaction between the adsorbed molecules and the underlying Bi/Cu(111) substrate as compared to the Ag(111) substrate. This allows us to efficiently enhance and tune the coupling of the tunneling current to localized excitations of the tip-sample junction, which in turn couple to radiative decay channels.

Light-sheet enhanced resolution of light field microscopy for rapid imaging of large volumes

NASA Astrophysics Data System (ADS)

Madrid Wolff, Jorge; Castro, Diego; Arbeláez, Pablo; Forero-Shelton, Manu

2018-02-01

Whole-brain imaging is challenging because it demands microscopes with high temporal and spatial resolution, which are often at odds, especially in the context of large fields of view. We have designed and built a light-sheet microscope with digital micromirror illumination and light-field detection. On the one hand, light sheets provide high resolution optical sectioning on live samples without compromising their viability. On the other hand, light field imaging makes it possible to reconstruct full volumes of relatively large fields of view from a single camera exposure; however, its enhanced temporal resolution comes at the expense of spatial resolution, limiting its applicability. We present an approach to increase the resolution of light field images using DMD-based light sheet illumination. To that end, we develop a method to produce synthetic resolution targets for light field microscopy and a procedure to correct the depth at which planes are refocused with rendering software. We measured the axial resolution as a function of depth and show a three-fold potential improvement with structured illumination, albeit by sacrificing some temporal resolution, also three-fold. This results in an imaging system that may be adjusted to specific needs without having to reassemble and realign it. This approach could be used to image relatively large samples at high rates.

Geometrical characterization of fluorescently labelled surfaces from noisy 3D microscopy data.

PubMed

Shelton, Elijah; Serwane, Friedhelm; Campàs, Otger

2018-03-01

Modern fluorescence microscopy enables fast 3D imaging of biological and inert systems alike. In many studies, it is important to detect the surface of objects and quantitatively characterize its local geometry, including its mean curvature. We present a fully automated algorithm to determine the location and curvatures of an object from 3D fluorescence images, such as those obtained using confocal or light-sheet microscopy. The algorithm aims at reconstructing surface labelled objects with spherical topology and mild deformations from the spherical geometry with high accuracy, rather than reconstructing arbitrarily deformed objects with lower fidelity. Using both synthetic data with known geometrical characteristics and experimental data of spherical objects, we characterize the algorithm's accuracy over the range of conditions and parameters typically encountered in 3D fluorescence imaging. We show that the algorithm can detect the location of the surface and obtain a map of local mean curvatures with relative errors typically below 2% and 20%, respectively, even in the presence of substantial levels of noise. Finally, we apply this algorithm to analyse the shape and curvature map of fluorescently labelled oil droplets embedded within multicellular aggregates and deformed by cellular forces. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Combined Multidimensional Microscopy as a Histopathology Imaging Tool.

PubMed

Shami, Gerald J; Cheng, Delfine; Braet, Filip

2017-02-01

Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 232: 249-256, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

DOE PAGES

McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; ...

2015-09-22

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presencemore » of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.« less

Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope.

PubMed

Mercatelli, Raffaella; Ratto, Fulvio; Centi, Sonia; Soria, Silvia; Romano, Giovanni; Matteini, Paolo; Quercioli, Franco; Pini, Roberto; Fusi, Franco

2013-10-21

In this paper we report on a new use for dark-field microscopy in order to retrieve two-dimensional maps of optical parameters of a thin sample such as a cryptograph, a histological section, or a cell monolayer. In particular, we discuss the construction of quantitative charts of light absorbance and scattering coefficients of a polyvinyl alcohol film that was embedded with gold nanorods and then etched using a focused mode-locked Ti:Sapphire oscillator. Individual pulses from this laser excite plasmonic oscillations of the gold nanorods, thus triggering plastic deformations of the particles and their environment, which are confined within a few hundred nm of the light focus. In turn, these deformations modify the light absorbance and scattering landscape, which can be measured with optical resolution in a dark-field microscope equipped with an objective of tuneable numerical aperture. This technique may prove to be valuable for various applications, such as the fast readout of optically encoded data or to model functional interactions between light and biological tissue at the level of cellular organelles, including the photothermolysis of cancer.

Ultrastructure of medial rectus muscles in patients with intermittent exotropia.

PubMed

Yao, J; Wang, X; Ren, H; Liu, G; Lu, P

2016-01-01

PURPOSE To study the ultrastructure of the medial rectus in patients with intermittent exotropia at different ages.PATIENTS AND METHODS The medial recti were harvested surgically from 20 patients with intermittent exotropia. Patients were divided into adolescent (age<18 years, n=10) and adult groups (age >18 years, n=10). The normal control group included five patients without strabismus and undergoing eye enucleation. Hematoxylin and eosin staining and transmission electron microscopy were used to visualize the medial recti. Western blot was used to determine the levels of myosin and actin.RESULTS Varying fiber thickness, atrophy, and misalignment of the medial recti were visualized under optical microscope in patients with exotropia. Electron microscopy revealed sarcomere destruction, myofilament disintegration, unclear dark and light bands, collagen proliferation, and fibrosis. The adolescent group manifested significantly higher levels of myosin and actin than the adult group (P<0.05).CONCLUSION Younger patients with intermittent exotropia show stronger contraction of the medial recti compared with older patients. Our findings suggest that childhood was the appropriate time for surgery as the benefit of the intervention was better than in adulthood.

Proximal design for a multimodality endoscope with multiphoton microscopy, optical coherence microscopy and visual modalities

NASA Astrophysics Data System (ADS)

Kiekens, Kelli C.; Talarico, Olivia; Barton, Jennifer K.

2018-02-01

A multimodality endoscope system has been designed for early detection of ovarian cancer. Multiple illumination and detection systems must be integrated in a compact, stable, transportable configuration to meet the requirements of a clinical setting. The proximal configuration presented here supports visible light navigation with a large field of view and low resolution, high resolution multiphoton microscopy (MPM), and high resolution optical coherence microscopy (OCM). All modalities are integrated into a single optical system in the endoscope. The system requires two light sources: a green laser for visible light navigation and a compact fiber based femtosecond laser for MPM and OCM. Using an inline wavelength division multiplexer, the two sources are combined into a single mode fiber. To accomplish OCM, a fiber coupler is used to separate the femtosecond laser into a reference arm and signal arm. The reflected reference arm and the signal from the sample are interfered and wavelength separated by a reflection grating and detected using a linear array. The MPM signal is collimated and goes through a series of filters to separate the 2nd and 3rd harmonics as well as twophoton excitation florescence (2PEF) and 3PEF. Each signal is independently detected on a photo multiplier tube and amplified. The visible light is collected by multiple high numerical aperture fibers at the endoscope tip which are bundled into one SMA adapter at the proximal end and connected to a photodetector. This integrated system design is compact, efficient and meets both optical and mechanical requirements for clinical applications.

Hue-saturation-density (HSD) model for stain recognition in digital images from transmitted light microscopy.

PubMed

van Der Laak, J A; Pahlplatz, M M; Hanselaar, A G; de Wilde, P C

2000-04-01

Transmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density. The usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels. In the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space. In the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose. Copyright 2000 Wiley-Liss, Inc.

Stability and degradation of organic photovoltaics fabricated, aged, and characterized by the ISOS 3 inter-laboratory collaboration

NASA Astrophysics Data System (ADS)

Tanenbaum, David M.; Hermenau, Martin; Voroshazi, Eszter; Lloyd, Matthew T.; Galagan, Yulia; Zimmermann, Birger; Hösel, Markus; Dam, Henrik F.; Jørgensen, Mikkel; Gevorgyan, Suren; Kudret, Suleyman; Maes, Wouter; Lutsen, Laurence; Vanderzande, Dirk; Würfel, Uli; Andriessen, Ronn; Rösch, Roland; Hoppe, Harald; Lira-Cantu, Monica; Teran-Escobar, Gerardo; Dupuis, Aurélie; Bussière, Pierre-Olivier; Rivaton, Agnès.; Uzunoglu, Gülsah Y.; Germack, David; Andreasen, Birgitta; Madsen, Morten V.; Norrman, Kion; Bundgaard, Eva; Krebs, Frederik C.

2012-09-01

Seven distinct sets (n >= 12) of state of the art organic photovoltaic devices were prepared by leading research laboratories in a collaboration planned at the Third International Summit on Organic Photovoltaic Stability (ISOS-3). All devices were shipped to DTU and characterized simultaneously up to 1830 h in accordance with established ISOS-3 protocols under three distinct illumination conditions: accelerated full sun simulation; low level indoor fluorescent lighting; and dark storage with daily measurement under full sun simulation. Three nominally identical devices were used in each experiment both to provide an assessment of the homogeneity of the samples and to distribute samples for a variety of post soaking analytical measurements at six distinct laboratories enabling comparison at various stages in the degradation of the devices. Characterization includes current-voltage curves, light beam induced current (LBIC) imaging, dark lock-in thermography (DLIT), photoluminescence (PL), electroluminescence (EL), in situ incident photon-to-electron conversion efficiency (IPCE), time of flight secondary ion mass spectrometry (TOF-SIMS), cross sectional electron microscopy (SEM), UV visible spectroscopy, fluorescence microscopy, and atomic force microscopy (AFM). Over 100 devices with more than 300 cells were used in the study. We present here design of the device sets, results both on individual devices and uniformity of device sets from the wide range of characterization methods applied at different stages of aging under the three illumination conditions. We will discuss how these data can help elucidate the degradation mechanisms as well as the benefits and challenges associated with the unprecedented size of the collaboration.

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Vos, Winnok H., E-mail: winnok.devos@uantwerpen.be; Cell Systems and Imaging Research Group, Department of Molecular Biotechnology, Ghent University, Ghent; Beghuin, Didier

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALMmore » ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.« less

High-resolution fiber-optic microendoscopy for in situ cellular imaging.

PubMed

Pierce, Mark; Yu, Dihua; Richards-Kortum, Rebecca

2011-01-11

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user. We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture, in excised and living animal tissues, and in human tissues in vivo. Users have reported the use of several different fluorescent contrast agents, including proflavine, benzoporphyrin-derivative monoacid ring A (BPD-MA), and fluoroscein, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

Diagnostic electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickersin, G.R.

1988-01-01

In this book the author presents a comprehensive reference text on diagnostic electron microscopy. Throughout the book he illustrates how ultrastructural identification can be helpful for the recognition of cell type and the identification of mechanisms of pathogenesis in various diseases. In addition to electron microscopy photographs, there are also numerous light microscopy photographs for comparison. This text presents the classification of neoplasms in the order and arrangement most familiar to the pathologist. Contents: Introduction; Diagram of a Normal Cell; Normal Cell Function; Embryology; Neoplasms; Infectious Agents; Metabolic Diseases; Renal Diseases; Skeletal Muscle and Peripheral Nerve Diseases; Index.

Preliminary studies on LED-activated pyropheophorbide-α methyl ester killing cisplatin-resistant ovarian carcinoma cells

NASA Astrophysics Data System (ADS)

Tan, Yong; Xu, Chuan Shan; Xia, Xin Shu; Yu, He Ping; Bai, Ding Qun; He, Yong; Xu, Jing; Wang, Ping; Wang, Xin Na; Leung, Albert Wing Nang

2009-05-01

In the present study, a novel LED source was applied for activating pyropheophorbids-a methyl ester (MPPa) in cisplatin-resistant ovarian cell line COC1/DDP cells. MPPa concentration was 2 μM and light energy from 0.125-8 J/cm2. Cytotoxicity was investigated 24 h using MTT reduction assay and light microscopy after treatment. Cellular ultrastructure was observed using transmission electron microscopy (TEM) and nuclear chromatin by fluorescent microscope with Hoechst33258 staining. MTT reduction assay showed that the cytotoxicity of LED-activated MPPa in the COC1/DDP cells increased along with the light dose of LED source and LED-activated MPPa resulted in light-dependent cytotoxicity. The observations from light microscopy reinforced the above results. TEM showed that necrotic cells with the disruption of karyotheca, karyorrhexis, and karyolysis of nucleus and apoptotic cells, especially the apoptotic body, can be seen post LED-activated MPPa. Hoechst33258 staining showed that condensation of chromatin and nuclear fragmentations could be found in many treated cells and some of them formed the structure of apoptotic bodies when COC1/DDP cells were exposed to 2 μM MPPa for 20 h and then 1 J/cm2 irradiation of LED source. The findings demonstrated that the novel LED source could efficiently activated MPPa and LED-activated MPPa could significantly kill cisplatin-resistant ovarian cell line COC1/DDP cells through two major pathways including necrosis and apoptosis, suggesting that LED is a novel and efficient light source and LED-activated MPPa might be potential therapeutic modality for treating cisplatin-resistant ovarian carcinoma.

Light, electron microscopic and immunohistochemical study of the effect of low-dose aspirin during the proestrus phase on rat endometrium in the preimplantation period.

PubMed

Ateş, Utku; Baka, Meral; Turgut, Mehmet; Uyanikgil, Yiğit; Ulker, Sibel; Yilmaz, Ozlem; Tavmergen, Erol; Yurtseven, Mine

2007-04-01

To evaluate structural alterations in rat endometrium at preimplantation following treatment with aspirin beginning from proestrus by light microscopy, electron microscopy and immunohistochemical techniques. Twenty rats were divided into control (n = 10) and experimental (n = 10) groups. Experimental rats were treated with low-dose aspirin daily (2 mg/kg/day) during estrus, beginning from the proestrus phase, mated at end of cycle and treated with aspirin. Untreated pregnant rats were the control group. Rats in both groups were sacrificed at the 84th pregnancy hour; the uterus was rapidly removed and dissected free of surrounding adipose tissue. Uteri specimens from nonpregnant rats were transferred into fixative solution and processed for light, electron microscopic and immunohistochemical study. Light and electron microscopy of endometrium from control rats conformed to mid-diestrus phase; endometrial histology of the aspirin-treated group conformed to late diestrus phase. The endometrial layer was significantly thicker in the aspirin-treated group compared to the untreated control group (p <0.001). No significant difference was found in vessel number between groups. Staining with alphaV integrin was more dense in the aspirin-treated group. Based on histologic findings, we suggest low-dose aspirin has positive effects on preparing endometrium before implantation.

Quantitative analysis with advanced compensated polarized light microscopy on wavelength dependence of linear birefringence of single crystals causing arthritis

NASA Astrophysics Data System (ADS)

Takanabe, Akifumi; Tanaka, Masahito; Taniguchi, Atsuo; Yamanaka, Hisashi; Asahi, Toru

2014-07-01

To improve our ability to identify single crystals causing arthritis, we have developed a practical measurement system of polarized light microscopy called advanced compensated polarized light microscopy (A-CPLM). The A-CPLM system is constructed by employing a conventional phase retardation plate, an optical fibre and a charge-coupled device spectrometer in a polarized light microscope. We applied the A-CPLM system to measure linear birefringence (LB) in the visible region, which is an optical anisotropic property, for tiny single crystals causing arthritis, i.e. monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD). The A-CPLM system performance was evaluated by comparing the obtained experimental data using the A-CPLM system with (i) literature data for a standard sample, MgF2, and (ii) experimental data obtained using an established optical method, high-accuracy universal polarimeter, for the MSUM. The A-CPLM system was found to be applicable for measuring the LB spectra of the single crystals of MSUM and CPPD, which cause arthritis, in the visible regions. We quantitatively reveal the large difference in LB between MSUM and CPPD crystals. These results demonstrate the usefulness of the A-CPLM system for distinguishing the crystals causing arthritis.

Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

NASA Astrophysics Data System (ADS)

Candeo, Alessia; Sana, Ilenia; Ferrari, Eleonora; Maiuri, Luigi; D'Andrea, Cosimo; Valentini, Gianluca; Bassi, Andrea

2016-05-01

Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability.

Prolactin-secreting pituitary adenoma in a man with gigantism: a case report.

PubMed

Peillon, F; Philippon, J; Brandi, A M; Fohanno, D; Laplane, D; Dubois, M P; Decourt, J

1979-12-01

A prolactin-secreting pituitary adenoma was removed trans-sphenoidally from a 37 years old man with gigantism (218 cm). Serum levels of prolactin (PRL) were elevated pre-operatively and decreased after administration of L-Dopa with no increase after TRH as is usually observed in PRL-secreting adenomas. Growth hormone (GH) and somatomedin serum levels were normal with no modification of GH after insulin hypoglycemia, oral glucose loading or L-Dopa. Morphological examination of the tumour demonstrated the presence of lactotrophs by light and electron microscopy and by immunofluorescense staining. No somatotrophs were found. In this unique case, the relationship between a PRL-secreting adenoma and gigantism is discussed.

Localization of nitric oxide synthase and NADPH-diaphorase in guinea pig and human cochleae.

PubMed

Ruan, R S; Leong, S K; Yeoh, K H

1997-01-01

The distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and nitric oxide synthase (NOS) in mammalian cochlea were studied at light and electron microscope levels by NADPH-d histochemistry and brain NOS (bNOS) immunohistochemistry. The cochleae from 15 albino guinea pigs were perilymphatically fixed with 2% periodate-lysine-paraformaldehyde, decalcified in 10% EDTA and processed for light and electron microscopy after NADPH-d or NOS staining in frozen and vibratome sections respectively. One human cochlea was available for light microscope examination of NADPH-d or bNOS stained sections. Light microscope results revealed that type I neurons and nerve fibers of the spiral ganglion cells were labeled by bNOS immunohistochemistry as well as NADPH-d histochemistry in both guinea pig and human cochleae. At subcellular level, NADPH-d reaction product was localized in the mitochondria of the neuronal cytoplasm and axoplasm and in the cytoplasm of the vascular endothelium. The immunoreaction products of bNOS were evenly distributed in the neuronal cytoplasm and axoplasm. Myelinated and unmyelinated fibers in the intraganglionic spiral bundle and the inner spiral and inner radial fibers below the inner hair cells were labeled for bNOS. The nerve endings below the outer hair cells were not stained. NOS immunoreaction product was also found in the outer hair cells, Schwann cells of myelinated nerve fibers, Deiter's cells, pillar cells and the tympanic lamina cells. No difference was found in the staining pattern of both NADPH-d and NOS reaction products between human and guinea pig cochleae at the light microscope level. The results suggest that NO plays an important role in the maintenance of auditory function in the mammal.

The 2015 super-resolution microscopy roadmap

NASA Astrophysics Data System (ADS)

Hell, Stefan W.; Sahl, Steffen J.; Bates, Mark; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J.; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Jakobs, Stefan; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J.; Eggeling, Christian; Klenerman, David; Willig, Katrin I.; Vicidomini, Giuseppe; Castello, Marco; Diaspro, Alberto; Cordes, Thorben

2015-11-01

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling

Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction

PubMed Central

Denker, Annette; Kröhnert, Katharina; Rizzoli, Silvio O

2009-01-01

The synaptic vesicles are organized in distinct populations or ‘pools’: the readily releasable pool (the first vesicles released upon stimulation), the recycling pool (which maintains release under moderate stimulation) and the reserve pool (which is called into action only upon strong, often unphysiological stimulation). A major question in the field is whether the pools consist of biochemically different vesicles or whether the pool tag is a spatial one (with the recycling vesicles found next to the release sites, and the reserve ones farther away). A strong and stable spatial segregation has been proposed in the last decade in the Drosophila larval neuromuscular junction – albeit based solely on light microscopy experiments. We have tested here this hypothesis using electron microscopy (EM) photoconversion. We found the recycling and reserve pools to be thoroughly intermixed at the EM level, indicating that spatial location is irrelevant for the functional properties of the vesicle. PMID:19403600

Multiscale 3D virtual dissections of 100-million-year-old flowers using X-ray synchrotron micro- and nanotomography.

PubMed

Moreau, Jean-David; Cloetens, Peter; Gomez, Bernard; Daviero-Gomez, Véronique; Néraudeau, Didier; Lafford, Tamzin A; Tafforeau, Paul

2014-02-01

A multiscale approach combining phase-contrast X-ray micro- and nanotomography is applied for imaging a Cretaceous fossil inflorescence in the resolution range from 0.75 μm to 50 nm. The wide range of scale views provides three-dimensional reconstructions from the external gross morphology of the inflorescence fragment to the finest exine sculptures of in situ pollen. This approach enables most of the characteristics usually observed under light microscopy, or with low magnification under scanning and transmission electron microscopy, to be obtained nondestructively. In contrast to previous tomography studies of fossil and extant flowers that used resolutions down to the micron range, we used voxels with a 50 nm side in local tomography scans. This high level of resolution enables systematic affinities of fossil flowers to be established without breaking or slicing specimens.

Confocal multispot microscope for fast and deep imaging in semicleared tissues

NASA Astrophysics Data System (ADS)

Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

2018-02-01

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Ahmed, Syeed Ehsan

Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

A polyphasic taxonomic approach in isolated strains of Cyanobacteria from thermal springs of Greece.

PubMed

Bravakos, Panos; Kotoulas, Georgios; Skaraki, Katerina; Pantazidou, Adriani; Economou-Amilli, Athena

2016-05-01

Strains of Cyanobacteria isolated from mats of 9 thermal springs of Greece have been studied for their taxonomic evaluation. A polyphasic taxonomic approach was employed which included: morphological observations by light microscopy and scanning electron microscopy, maximum parsimony, maximum likelihood and Bayesian analysis of 16S rDNA sequences, secondary structural comparisons of 16S-23S rRNA Internal Transcribed Spacer sequences, and finally environmental data. The 17 cyanobacterial isolates formed a diverse group that contained filamentous, coccoid and heterocytous strains. These included representatives of the polyphyletic genera of Synechococcus and Phormidium, and the orders Oscillatoriales, Spirulinales, Chroococcales and Nostocales. After analysis, at least 6 new taxa at the genus level provide new evidence in the taxonomy of Cyanobacteria and highlight the abundant diversity of thermal spring environments with many potential endemic species or ecotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

Electron microscopic demonstration of hormone-producing metastatic carcinoma of the choroid from the bronchus.

PubMed

Amemiya, T; Nomura, S

1975-01-01

Clinical, laboratory and pathological findings of a patient in bronchial carcinoma with choroidal metastasis were presented. X-ray examination of the chest suggested the tumor shadow in the posterior segmental bronchus of the right upper lobe of the lung (r-B2b), while funduscopy and fluorescein angiography revealed the presence of choroidal tumor. ACTH levels in tumor tissues at autopsy and in serum were measured and definitely demonstrated and elevated. Histopathologically, the primary lesion was r-B2b and diagnosed as a mucocellular type of adenocarcinoma. The choroidal lesion was metastatic carcinoma. Electron microscopic examination of the choroidal lesion reembedded for electron microscopy from celloidin-embedded materials for light microscopy could reveal the presence of characteristic cytoplasmic granules referred to as neurosecretory-type granules. It is extremely rare that a hormone-producing metastatic carcinoma of the choroid from the bronchus has been proved.

Development of imaging techniques to study the pathogenesis of biosafety level 2/3 infectious agents

PubMed Central

Rella, Courtney E.; Ruel, Nancy; Eugenin, Eliseo A.

2015-01-01

Despite significant advances in microbiology and molecular biology over the last decades, several infectious diseases remain global concerns, resulting in the death of millions of people worldwide each year. According to the Center for Disease Control (CDC) in 2012, there were 34 million people infected with HIV, 8.7 million new cases of tuberculosis, 500 million cases of hepatitis, and 50–100 million people infected with dengue. Several of these pathogens, despite high incidence, do not have reliable clinical detection methods. New or improved protocols have been generated to enhance detection and quantitation of several pathogens using high-end microscopy (light, confocal, and STORM microscopy) and imaging software. In the current manuscript, we discuss these approaches and the theories behind these methodologies. Thus, advances in imaging techniques will open new possibilities to discover therapeutic interventions to reduce or eliminate the devastating consequences of infectious diseases. PMID:24990818

Redescription of four species of Mehdiella from Testudinidae, with a key to the species and discussion on the relationships among the species of this genus.

PubMed

Bouamer, S; Morand, S; Kara, M

2003-12-01

Four species of the genus Mehdiella Seurat, 1918 are redescribed: M. cristata Petter, 1966 and M. stylosa dollfusi Petter, 1966, parasite of Pyxix arachnoides Bell, 1827 from Madagascar, M. s. stylosa (Thapar, 1925) and M. uncinata (Drasche, 1884), parasite of Testudo graeca Linneaus, 1758, Testudo hermanni Gmelin, 1789 and Testudo horsfieldii (Gray, 1844) from Palaearctic region. Light microscopy and scanning electron microscopy (SEM) studies revealed new informations on the morphology of these species. On the basis of this morphological study, the sub-species Mehdiella stylosa dollfusi and M. s. stylosa are raised to level of species. The position of Mehdiella cristata among the species of the genus Mehdiella and the relationships among the species of the genus Mehdiella are discussed. A key to the eight valid species Mehdiella is given.

The proteomic profile of hair damage.

PubMed

Sinclair, R; Flagler, M J; Jones, L; Rufaut, N; Davis, M G

2012-06-01

Monilethrix is a congenital hair shaft disorder with associated fragility. Many of the changes seen in monilethrix hair on light microscopy and scanning electron microscopy are also seen in hair weathering and cosmetic damage to hair. We used monilethrix as a model to investigate the relationship between hair protein structure and hair strength and resistance to cosmetic insult. We applied proteomic techniques to identify novel peptide damage markers for chemical oxidative damage to hair. The findings suggest that specific sites in the protein structure of hair are targeted during oxidative damage from bleaching, a unique insight into how chemical damage compromises the structural integrity of the hair shaft at the molecular level. Applying proteomics to the study of congenital and acquired hair shaft disorders can deliver new insights into hair damage and novel strategies to strengthen hair. © 2012 The Authors. BJD © 2012 British Association of Dermatologists.

Strain and lattice orientation distribution in SiN/Ge complementary metal–oxide–semiconductor compatible light emitting microstructures by quick x-ray nano-diffraction microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chahine, G. A.; Schülli, T. U.; Zoellner, M. H.

2015-02-16

This paper presents a study of the spatial distribution of strain and lattice orientation in CMOS-fabricated strained Ge microstripes using high resolution x-ray micro-diffraction. The recently developed model-free characterization tool, based on a quick scanning x-ray diffraction microscopy technique can image strain down to levels of 10{sup −5} (Δa/a) with a spatial resolution of ∼0.5 μm. Strain and lattice tilt are extracted using the strain and orientation calculation software package X-SOCS. The obtained results are compared with the biaxial strain distribution obtained by lattice parameter-sensitive μ-Raman and μ-photoluminescence measurements. The experimental data are interpreted with the help of finite element modelingmore » of the strain relaxation dynamics in the investigated structures.« less

[The morphofunctional changes in the rat lung tissue during simulation of acute fatal poisoning with household gas].

PubMed

Kalinina, E Iu; Iagmurov, O D

2014-01-01

The methods of light microscopy, immunohistochemistry, and electron microscopy were employed to study the morphofunctional changes in epithelium of bronchial and respiratory segments of the rat lungs used as models of acute fatal poisoning with household gas. It was shown that this toxic effect induces the pathological process involving all the elements of the epithelial layer in the bronchial and respiratory segments of the lungs of experimental animals. At the ultrastructural level, mitochondria and endoplasmic reticulum structures are affected, with the death of epithelial cells leading to the damage of the aerohematic barrier. The toxic effect of the gaseous mixture on the membranes causes the destruction of various elements of the epithelial layer. The results of this study help to understand the mechanisms of death in the case of acute fatal poisoning with household gas.

Tegumental histological effects of Mirazid(®) and myrrh volatile oil on adult Fasciola gigantica.

PubMed

Massoud, Ahmad Mohamed; Shalaby, Hatem Abdel Mawgoud; El Khateeb, Rabab Mohamed; Mahmoud, Mona Said; Kutkat, Mohamed Abdel Aziz

2013-06-01

To evaluated the histological changes within the tegument of adult Fasciola gigantica (F. gigantica) that led to the gross changes that were visible externally. The effects of oleoresin extract of myrrh (Mirazid(®)), myrrh volatile oil and triclabendazole sulphoxide (reference drug) on the tegumental structure of adult F. gigantica following treatment in vitro had been determined by light microscopy. The internal changes in the tegument observed in this study were compatible with surface changes seen in the previous scanning electron microscopy study, using the same drugs. The swelling of tegumental syncytium was a particular feature of their action, but its level was much greater with myrrh volatile oil, in which vacuolization of the tegument and loss of spines were observed. The present study demonstrated the fasciocidal properties of Mirazid(®) oleoresin extract, and it might be possible to reinforce its fasciocidal activity by increasing its content of myrrh volatile oil.

Quantum correlation enhanced super-resolution localization microscopy enabled by a fibre bundle camera

PubMed Central

Israel, Yonatan; Tenne, Ron; Oron, Dan; Silberberg, Yaron

2017-01-01

Despite advances in low-light-level detection, single-photon methods such as photon correlation have rarely been used in the context of imaging. The few demonstrations, for example of subdiffraction-limited imaging utilizing quantum statistics of photons, have remained in the realm of proof-of-principle demonstrations. This is primarily due to a combination of low values of fill factors, quantum efficiencies, frame rates and signal-to-noise characteristic of most available single-photon sensitive imaging detectors. Here we describe an imaging device based on a fibre bundle coupled to single-photon avalanche detectors that combines a large fill factor, a high quantum efficiency, a low noise and scalable architecture. Our device enables localization-based super-resolution microscopy in a non-sparse non-stationary scene, utilizing information on the number of active emitters, as gathered from non-classical photon statistics. PMID:28287167

An easy-to-implement filter for separating photo-excited signals from topography in scanning tunneling microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Kangkang; Rosenmann, Daniel; Holt, Martin

2013-06-15

In order to achieve elemental and chemical sensitivity in scanning tunneling microscopy (STM), synchrotron x-rays have been applied to excite core-level electrons during tunneling. The x-ray photo-excitations result in tip currents that are superimposed onto conventional tunneling currents. While carrying important physical information, the varying x-ray induced currents can destabilize the feedback loop causing it to be unable to maintain a constant tunneling current, sometimes even causing the tip to retract fully or crash. In this paper, we report on an easy-to-implement filter circuit that can separate the x-ray induced currents from conventional tunneling currents, thereby allowing simultaneous measurements ofmore » topography and chemical contrasts. The filter and the schematic presented here can also be applied to other variants of light-assisted STM such as laser STM.« less

Climbing Bloom's taxonomy pyramid: Lessons from a graduate histology course.

PubMed

Zaidi, Nikki B; Hwang, Charles; Scott, Sara; Stallard, Stefanie; Purkiss, Joel; Hortsch, Michael

2017-09-01

Bloom's taxonomy was adopted to create a subject-specific scoring tool for histology multiple-choice questions (MCQs). This Bloom's Taxonomy Histology Tool (BTHT) was used to analyze teacher- and student-generated quiz and examination questions from a graduate level histology course. Multiple-choice questions using histological images were generally assigned a higher BTHT level than simple text questions. The type of microscopy technique (light or electron microscopy) used for these image-based questions did not result in any significant differences in their Bloom's taxonomy scores. The BTHT levels for teacher-generated MCQs correlated positively with higher discrimination indices and inversely with the percent of students answering these questions correctly (difficulty index), suggesting that higher-level Bloom's taxonomy questions differentiate well between higher- and lower-performing students. When examining BTHT scores for MCQs that were written by students in a Multiple-Choice Item Development Assignment (MCIDA) there was no significant correlation between these scores and the students' ability to answer teacher-generated MCQs. This suggests that the ability to answer histology MCQs relies on a different skill set than the aptitude to construct higher-level Bloom's taxonomy questions. However, students significantly improved their average BTHT scores from the midterm to the final MCIDA task, which indicates that practice, experience and feedback increased their MCQ writing proficiency. Anat Sci Educ 10: 456-464. © 2017 American Association of Anatomists. © 2017 American Association of Anatomists.

4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)

PubMed Central

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

Photodynamic inactivation of pathogenic species Pseudomonas aeruginosa and Candida albicans with lutetium (III) acetate phthalocyanines and specific light irradiation.

PubMed

Mantareva, Vanya; Kussovski, Vesselin; Durmuş, Mahmut; Borisova, Ekaterina; Angelov, Ivan

2016-11-01

Photodynamic inactivation (PDI) is a light-associated therapeutic approach suitable for treatment of local acute infections. The method is based on specific light-activated compound which by specific irradiation and in the presence of molecular oxygen produced molecular singlet oxygen and other reactive oxygen species, all toxic for pathogenic microbial cells. The study presents photodynamic impact of two recently synthesized water-soluble cationic lutetium (III) acetate phthalocyanines (LuPc-5 and LuPc-6) towards two pathogenic strains, namely, the Gram-negative bacterium Pseudomonas aeruginosa and a fungus Candida albicans. The photodynamic effect was evaluated for the cells in suspensions and organized in 48-h developed biofilms. The relatively high levels of uptakes of LuPc-5 and LuPc-6 were determined for fungal cells compared to bacterial cells. The penetration depths and distribution of both LuPcs into microbial biofilms were investigated by means of confocal fluorescence microscopy. The photoinactivation efficiency was studied for a wide concentration range (0.85-30 μM) of LuPc-5 and LuPc-6 at a light dose of 50 J cm -2 from red light-emitting diode (LED; 665 nm). The PDI study on microbial biofilms showed incomplete photoinactivation (<3 logs) for the used gentle drug-light protocol.

Micro-CT scouting for transmission electron microscopy of human tissue specimens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morales, A. G.; Stempinski, E. S.; XIAO, X.

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. Here, we describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 m resolution was used to determine the location of patches of themore » mucous membrane in osmium-stained human nasal scraping samples. Furthermore, once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation. Lay description Electron microscopy provides very high levels of detail in a small area, and thus the question of where to look in an opaque sample, such as a stained tissue specimen, needs to be answered by sectioning the sample in small steps and examining the sections under a light microscope, until the region of interest is found. The search process can be lengthy and labor intensive, especially for a study involving a large number of samples. Small areas of interest can be missed in the process if not enough regions are examined. We also describe a method to directly locate the region of interest within a whole sample using micro-CT imaging, bypassing the need of blindly sectioning. Micro-CT enables locating the region within 3D space; this information provides a guide for sectioning the sample to expose that precise location for high resolution electron microscopy imaging. In a human tissue specimen study, this method considerably reduced the time and labor of the search process.« less

Micro-CT scouting for transmission electron microscopy of human tissue specimens

DOE PAGES

Morales, A. G.; Stempinski, E. S.; XIAO, X.; ...

2016-02-08

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. Here, we describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 m resolution was used to determine the location of patches of themore » mucous membrane in osmium-stained human nasal scraping samples. Furthermore, once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation. Lay description Electron microscopy provides very high levels of detail in a small area, and thus the question of where to look in an opaque sample, such as a stained tissue specimen, needs to be answered by sectioning the sample in small steps and examining the sections under a light microscope, until the region of interest is found. The search process can be lengthy and labor intensive, especially for a study involving a large number of samples. Small areas of interest can be missed in the process if not enough regions are examined. We also describe a method to directly locate the region of interest within a whole sample using micro-CT imaging, bypassing the need of blindly sectioning. Micro-CT enables locating the region within 3D space; this information provides a guide for sectioning the sample to expose that precise location for high resolution electron microscopy imaging. In a human tissue specimen study, this method considerably reduced the time and labor of the search process.« less

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

Prediction of Prostate Cancer Recurrence Using Quantitative Phase Imaging

NASA Astrophysics Data System (ADS)

Sridharan, Shamira; Macias, Virgilia; Tangella, Krishnarao; Kajdacsy-Balla, André; Popescu, Gabriel

2015-05-01

The risk of biochemical recurrence of prostate cancer among individuals who undergo radical prostatectomy for treatment is around 25%. Current clinical methods often fail at successfully predicting recurrence among patients at intermediate risk for recurrence. We used a label-free method, spatial light interference microscopy, to perform localized measurements of light scattering in prostatectomy tissue microarrays. We show, for the first time to our knowledge, that anisotropy of light scattering in the stroma immediately adjoining cancerous glands can be used to identify patients at higher risk for recurrence. The data show that lower value of anisotropy corresponds to a higher risk for recurrence, meaning that the stroma adjoining the glands of recurrent patients is more fractionated than in non-recurrent patients. Our method outperformed the widely accepted clinical tool CAPRA-S in the cases we interrogated irrespective of Gleason grade, prostate-specific antigen (PSA) levels and pathological tumor-node-metastasis (pTNM) stage. These results suggest that QPI shows promise in assisting pathologists to improve prediction of prostate cancer recurrence.

HANFORD WASTE MINERALOGY REFERENCE REPORT

DOE Office of Scientific and Technical Information (OSTI.GOV)

DISSELKAMP RS

2010-06-29

This report lists the observed mineral phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports that used experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases observed in Hanford waste.

HANFORD WASTE MINEROLOGY REFERENCE REPORT

DOE Office of Scientific and Technical Information (OSTI.GOV)

DISSELKAMP RS

2010-06-18

This report lists the observed mineral phase phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports using experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases present observed in Hanford waste.

Remineralization Potential of Three Tooth Pastes on Enamel Caries.

PubMed

Singhal, Rajnish K; Rai, Balwant

2017-08-15

Different formulations of dentifrices exist in the market. Usually, single toothpaste is used by all family members including children. There is a big concern of fluoride ingestion with the toothpaste containing high fluoride content in children. Recently, new toothpaste (including toothpaste) with remineralization potential without fluoride content has been formulated. There is an urgent need to compare remineralization potential of this new formulation with the exiting dentifrices. Therefore, the present study has been undertaken to assess and compare the remineralization potential of three dentifrices with different compositions on artificially induced carious lesions in vitro by using scanning electron microscopy and polarised light microscopy. The present in vitro study was conducted on 21 healthy extracted primary central incisor teeth surfaces, which were divided into three groups and were treated by three different dentifrices. Artificial demineralization was followed by remineralization using dentifrice slurry as per the group distribution. All the samples were studied for remineralization by using scanning electron microscopy and polarised light microscopy. Data were analysed using SPSS version 11 software. A significant difference was found between the remineralization potential of incudent toothpaste and other toothpaste groups based on the analysis of polarised light microscopy and stereomicroscope. The remineralizing ability of incudent toothpaste for artificial enamel lesions was found to be significantly higher than that of Colgate® and Crest toothpaste. The limitations of this study include, being a short term study, low sample size and in vitro experiment. incudent toothpaste has exhibited a higher remineralizing potential as compared to fluoride based toothpaste in our study.

Two-photon confocal microscopy in wound healing

NASA Astrophysics Data System (ADS)

Navarro, Fernando A.; So, Peter T. C.; Driessen, Antoine; Kropf, Nina; Park, Christine S.; Huertas, Juan C.; Lee, Hoon B.; Orgill, Dennis P.

2001-04-01

Advances in histopathology and immunohistochemistry have allowed for precise microanatomic detail of tissues. Two Photon Confocal Microscopy (TPCM) is a new technology useful in non-destructive analysis of tissue. Laser light excites the natural florophores, NAD(P)H and NADP+ and the scattering patterns of the emitted light are analyzed to reconstruct microanatomic features. Guinea pig skin was studied using TPCM and skin preparation methods including chemical depilation and tape striping. Results of TPCM were compared with conventional hematoxylin and eosin microscopy. Two-dimensional images were rendered from the three dimensional reconstructions. Images of deeper layers including basal cells and the dermo-epidermal junction improved after removing the stratum corneum with chemical depilation or tape stripping. TCPM allows good resolution of corneocytes, basal cells and collagen fibers and shows promise as a non-destructive method to study wound healing.

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device.

PubMed

Cheng, Li-Chung; Lien, Chi-Hsiang; Da Sie, Yong; Hu, Yvonne Yuling; Lin, Chun-Yu; Chien, Fan-Ching; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2014-08-01

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

Correlative fractography: combining scanning electron microscopy and light microscopes for qualitative and quantitative analysis of fracture surfaces.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-04-01

Correlative fractography is a new expression proposed here to describe a new method for the association between scanning electron microscopy (SEM) and light microscopy (LM) for the qualitative and quantitative analysis of fracture surfaces. This article presents a new method involving the fusion of one elevation map obtained by extended depth from focus reconstruction from LM with exactly the same area by SEM and associated techniques, as X-ray mapping. The true topographic information is perfectly associated to local fracture mechanisms with this new technique, presented here as an alternative to stereo-pair reconstruction for the investigation of fractured components. The great advantage of this technique resides in the possibility of combining any imaging methods associated with LM and SEM for the same observed field from fracture surface.

Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

PubMed

González-Robles, Arturo; Lares-Villa, Fernando; Lares-Jiménez, Luis Fernando; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Martínez-Palomo, Adolfo

2015-10-01

Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

Going "open" with mesoscopy: a new dimension on multi-view imaging.

PubMed

Gualda, Emilio; Moreno, Nuno; Tomancak, Pavel; Martins, Gabriel G

2014-03-01

OpenSPIM and OpenSpinMicroscopy emerged as open access platforms for Light Sheet and Optical Projection Imaging, often called as optical mesoscopy techniques. Both projects can be easily reproduced using comprehensive online instructions that should foster the implementation and further development of optical imaging techniques with sample rotation control. This additional dimension in an open system offers the possibility to make multi-view microscopy easily modified and will complement the emerging commercial solutions. Furthermore, it is deeply based on other open platforms such as MicroManager and Arduino, enabling development of tailored setups for very specific biological questions. In our perspective, the open access principle of OpenSPIM and OpenSpinMicroscopy is a game-changer, helping the concepts of light sheet and optical projection tomography (OPT) to enter the mainstream of biological imaging.

Oscheius wisconsinensis n. sp. (Nematoda: Rhabditidae), a potential entomopathogenic nematode from the marshlands of Wisconsin

USDA-ARS?s Scientific Manuscript database

Oscheius wisconsinensis n. sp. (Rhabditidae) was recovered through the Galleria bait method from a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit...

Plane wave analysis of coherent holographic image reconstruction by phase transfer (CHIRPT).

PubMed

Field, Jeffrey J; Winters, David G; Bartels, Randy A

2015-11-01

Fluorescent imaging plays a critical role in a myriad of scientific endeavors, particularly in the biological sciences. Three-dimensional imaging of fluorescent intensity often requires serial data acquisition, that is, voxel-by-voxel collection of fluorescent light emitted throughout the specimen with a nonimaging single-element detector. While nonimaging fluorescence detection offers some measure of scattering robustness, the rate at which dynamic specimens can be imaged is severely limited. Other fluorescent imaging techniques utilize imaging detection to enhance collection rates. A notable example is light-sheet fluorescence microscopy, also known as selective-plane illumination microscopy, which illuminates a large region within the specimen and collects emitted fluorescent light at an angle either perpendicular or oblique to the illumination light sheet. Unfortunately, scattering of the emitted fluorescent light can cause blurring of the collected images in highly turbid biological media. We recently introduced an imaging technique called coherent holographic image reconstruction by phase transfer (CHIRPT) that combines light-sheet-like illumination with nonimaging fluorescent light detection. By combining the speed of light-sheet illumination with the scattering robustness of nonimaging detection, CHIRPT is poised to have a dramatic impact on biological imaging, particularly for in vivo preparations. Here we present the mathematical formalism for CHIRPT imaging under spatially coherent illumination and present experimental data that verifies the theoretical model.

Bidomain structures formed in lithium niobate and lithium tantalate single crystals by light annealing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubasov, I. V., E-mail: kubasov.ilya@gmail.com; Kislyuk, A. M.; Bykov, A. S.

The bidomain structures produced by light external heating in z-cut lithium niobate and lithium tantalate single crystals are formed and studied. Interdomain regions about 200 and 40 μm wide in, respectively, LiNbO{sub 3} and LiTaO{sub 3} bidomain crystals are visualized and studied by optical microscopy and piezoresponse force microscopy. Extended chains and lines of domains in the form of thin layers with a width less than 10 μm in volume, which penetrate the interdomain region and spread over distances of up to 1 mm, are found.

Two-photon excitation fluorescence bioassays.

PubMed

Hänninen, Pekka; Soukka, Jori; Soini, Juhani T

2008-01-01

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the "renaissance" of light microscopy that started in the 1980s. The technique derives its advantages from the biologically "smooth" wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation-based assay system.

Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis.

PubMed

Smith, Benjamin; Li, Jianfang; Metruccio, Matteo; Wan, Stephanie; Evans, David; Fleiszig, Suzanne

2018-04-20

A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples.

Biophysical mechanism of transient retinal phototropism in rod photoreceptors.

PubMed

Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Gai, Shaoyan; Yao, Xincheng

2016-02-13

Oblique light stimulation evoked transient retinal phototropism (TRP) has been recently detected in frog and mouse retinas. High resolution microscopy of freshly isolated retinas indicated that the TRP is predominated by rod photoreceptors. Comparative confocal microscopy and optical coherence tomography (OCT) revealed that the TRP predominantly occurred from the photoreceptor outer segment (OS). However, biophysical mechanism of rod OS change is still unknown. In this study, frog retinal slices, which open a cross section of retinal photoreceptor and other functional layers, were used to test the effect of light stimulation on rod OS. Near infrared light microscopy was employed to monitor photoreceptor changes in retinal slices stimulated by a rectangular-shaped visible light flash. Rapid rod OS length change was observed after the stimulation delivery. The magnitude and direction of the rod OS change varied with the position of the rods within the stimulated area. In the center of stimulated region the length of the rod OS shrunk, while in the peripheral region the rod OS tip swung towards center region in the plane perpendicular to the incident stimulus light. Our experimental result and theoretical analysis suggest that the observed TRP may reflect unbalanced disc-shape change due to localized pigment bleaching. Further investigation is required to understand biochemical mechanism of the observed rod OS kinetics. Better study of the TRP may provide a noninvasive biomarker to enable early detection of age-related macular degeneration (AMD) and other diseases that are known to produce retinal photoreceptor dysfunctions.

Biophysical mechanism of transient retinal phototropism in rod photoreceptors

NASA Astrophysics Data System (ADS)

Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Gai, Shaoyan; Yao, Xincheng

2016-03-01

Oblique light stimulation evoked transient retinal phototropism (TRP) has been recently detected in frog and mouse retinas. High resolution microscopy of freshly isolated retinas indicated that the TRP is predominated by rod photoreceptors. Comparative confocal microscopy and optical coherence tomography (OCT) revealed that the TRP predominantly occurred from the photoreceptor outer segment (OS). However, biophysical mechanism of rod OS change is still unknown. In this study, frog retinal slices, which open a cross section of retinal photoreceptor and other functional layers, were used to test the effect of light stimulation on rod OS. Near infrared light microscopy was employed to monitor photoreceptor changes in retinal slices stimulated by a rectangular-shaped visible light flash. Rapid rod OS length change was observed after the stimulation delivery. The magnitude and direction of the rod OS change varied with the position of the rods within the stimulated area. In the center of stimulated region the length of the rod OS shrunk, while in the peripheral region the rod OS tip swung towards center region in the plane perpendicular to the incident stimulus light. Our experimental result and theoretical analysis suggest that the observed TRP may reflect unbalanced disc-shape change due to localized pigment bleaching. Further investigation is required to understand biochemical mechanism of the observed rod OS kinetics. Better study of the TRP may provide a noninvasive biomarker to enable early detection of age-related macular degeneration (AMD) and other diseases that are known to produce retinal photoreceptor dysfunctions.

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator.

PubMed

Shao, Yonghong; Qin, Wan; Liu, Honghai; Qu, Junle; Peng, Xiang; Niu, Hanben; Gao, Bruce Z

2012-07-01

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.

Quantitative luminescence imaging system

DOEpatents

Erwin, David N.; Kiel, Johnathan L.; Batishko, Charles R.; Stahl, Kurt A.

1990-01-01

The QLIS images and quantifies low-level chemiluminescent reactions in an electromagnetic field. It is capable of real time nonperturbing measurement and simultaneous recording of many biochemical and chemical reactions such as luminescent immunoassays or enzyme assays. The system comprises image transfer optics, a low-light level digitizing camera with image intensifying microchannel plates, an image process or, and a control computer. The image transfer optics may be a fiber image guide with a bend, or a microscope, to take the light outside of the RF field. Output of the camera is transformed into a localized rate of cumulative digitalized data or enhanced video display or hard-copy images. The system may be used as a luminescent microdosimetry device for radiofrequency or microwave radiation, as a thermal dosimeter, or in the dosimetry of ultra-sound (sonoluminescence) or ionizing radiation. It provides a near-real-time system capable of measuring the extremely low light levels from luminescent reactions in electromagnetic fields in the areas of chemiluminescence assays and thermal microdosimetry, and is capable of near-real-time imaging of the sample to allow spatial distribution analysis of the reaction. It can be used to instrument three distinctly different irradiation configurations, comprising (1) RF waveguide irradiation of a small Petri-dish-shaped sample cell, (2) RF irradiation of samples in a microscope for the microscopie imaging and measurement, and (3) RF irradiation of small to human body-sized samples in an anechoic chamber.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Minimal resin embedding of multicellular specimens for targeted FIB-SEM imaging.

PubMed

Schieber, Nicole L; Machado, Pedro; Markert, Sebastian M; Stigloher, Christian; Schwab, Yannick; Steyer, Anna M

2017-01-01

Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a Multicomponent Traditional Herbal Medicine by HPLC-MS and Electron and Light Microscopy.

PubMed

Liu, Ju-Han; Cheng, Yung-Yi; Hsieh, Chen-Hsi; Tsai, Tung-Hu

2017-12-15

Commercial pharmaceutical herbal products have enabled people to take traditional Chinese medicine (TCM) in a convenient and accessible form. However, the quantity and quality should be additionally inspected. To address the issue, a combination of chemical and physical inspection methods were developed to evaluate the amount of an herbal formula, Xiang-Sha-Liu-Jun-Zi-Tang (XSLJZT), in clinical TCM practice. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method with electrospray ionization was developed to measure the herbal biomarkers of guanosine, atractylenolide III, glycyrrhizic acid, dehydrocostus lactone, hesperidin, and oleanolic acid from XSLJZT. Scanning electron microscopy (SEM) photographs and light microscopy photographs with Congo red and iodine-KI staining were used to identify the cellulose fibers and starch content. Furthermore, solubility analysis, swelling power test, and crude fiber analysis were contributed to measure the starch additive in pharmaceutical products. The results demonstrated large variations in the chemical components of different pharmaceutical brands. The SEM photographs revealed that the starch was oval, smooth, and granular, and that the raw herbal powder appears stripy, stretched, and filiform. The stained light microscopy photographs of all of the pharmaceutical products showed added starch and raw herbal powder as extenders. The developed chemical and physical methods provide a standard operating procedure for the quantity control of the herbal pharmaceutical products of XSLJZT.

Elastomeric photo-actuators and their investigation by confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Czaniková, Klaudia; Ilčíková, Markéta; Krupa, Igor; Mičušík, Matej; Kasák, Peter; Pavlova, Ewa; Mosnáček, Jaroslav; Chorvát, Dušan, Jr.; Omastová, Mária

2013-10-01

The photo-actuation behavior of nanocomposites based on ethylene-vinylacetate copolymer (EVA) and styrene-isoprene-styrene (SIS) block copolymer filled with well-dispersed and modified multiwalled carbon nanotubes (MWCNTs) is discussed in this paper. The nanocomposites were prepared by casting from solution. To improve the dispersion of the MWCNTs in EVA, the MWCNT surface was modified with a non-covalent surfactant, cholesteryl 1-pyrenecarboxylate (PyChol). To prepare SIS nanocomposites, the MWCNT surface was covalently modified with polystyrene chains. The good dispersion of the filler was confirmed by transmission electron microscopy (TEM). Special, custom-made punch/die molds were used to create a Braille element (BE)-like shape, which under shear forces induces a uniaxial orientation of the MWCNTs within the matrix. The uniaxial orientation of MWCNTs is an essential precondition to ensure the photo-actuating behavior of MWCNTs in polymeric matrices. The orientation of the MWCNTs within the matrices was examined by scanning electron microscopy (SEM). Nanocomposite BEs were illuminated from the bottom by a red light-emitting diode (LED), and the photo-actuation was investigated by confocal laser scanning microscopy (CLSM). When the BEs were exposed to light, a temporary increase in the height of the element was detected. This process was observed to be reversible: after switching off the light, the BEs returned to their original shape and height.

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy.

PubMed

Zhao, Ziqing W; Roy, Rahul; Gebhardt, J Christof M; Suter, David M; Chapman, Alec R; Xie, X Sunney

2014-01-14

Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of "transcription factories." Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.

Identification of Foreign Particles in Human Tissues using Raman Microscopy.

PubMed

Campion, Alan; Smith, Kenneth J; Fedulov, Alexey V; Gregory, David; Fan, Yuwei; Godleski, John J

2018-06-12

The precise identification of foreign particles in tissue for patient care and research has been studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction. The goal of this study was to unambiguously identify particles in tissues using a combina-tion of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicron spatial resolution. We designed a model system of stained and unstained cells that contained birefringent talc particles, and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra ob-tained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely as-signed to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micron in diameter. Among commonly used coverslip mounting media, Cytoseal 60 is recommended; Permount was unacceptable due to intense background interference. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

Advanced 3D Optical Microscopy in ENS Research.

PubMed

Vanden Berghe, Pieter

2016-01-01

Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall.

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions

PubMed Central

Ip, Yuen K.; Hiong, Kum C.; Goh, Enan J. K.; Boo, Mel V.; Choo, Celine Y. L.; Ching, Biyun; Wong, Wai P.; Chew, Shit F.

2017-01-01

Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam. PMID:29066980

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions.

PubMed

Ip, Yuen K; Hiong, Kum C; Goh, Enan J K; Boo, Mel V; Choo, Celine Y L; Ching, Biyun; Wong, Wai P; Chew, Shit F

2017-01-01

Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca 2+ to, and simultaneously an augmented removal of H + from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca 2+ -ATPase ( PMCA ) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa . The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca 2+ in exchange for H + . More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA /PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

PubMed

Veys, Pascal; Planchon, Viviane; Colbert, Ruairi; Cruz, Clara; Frick, Geneviève; Ioannou, Ioannis; Marchis, Daniela; Nordkvist, Erik; Paradies-Severin, Inge; Pohto, Arja; Weiss, Roland; Baeten, Vincent; Berben, Gilbert

2017-08-01

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

Stability of cp-Ti and Ti-6Al-4V alloy for dental implants as a function of saliva pH - an electrochemical study.

PubMed

Barão, Valentim A R; Mathew, Mathew T; Assunção, Wirley Gonçalves; Yuan, Judy Chia-Chun; Wimmer, Markus A; Sukotjo, Cortino

2012-09-01

To investigate the role of different levels of pH of artificial saliva under simulated oral environment on the corrosion behavior of commercially pure titanium (cp-Ti) and Ti-6Al-4V alloy. Special attention is given to understand the changes in corrosion kinetics and surface characterization of Ti by using electrochemical impedance spectroscopy (EIS). Fifty-four Ti disks (15-mm diameter, 2-mm thickness) were divided into six groups (n = 9) as a function of saliva pH (3, 6.5, and 9) and Ti type. Samples were mechanically polished using standard metallographic procedures. Standard electrochemical tests, such as open circuit potential, EIS, and potentiodynamic tests were conducted in a controlled environment. Data were evaluated by two-way ANOVA, Tukey multiple comparison test, and independent t-test (α = 0.05). Ti surfaces were examined using white-light-interferometry microscopy and scanning electron microscopy (SEM). Saliva pH level significantly affected the corrosion behavior of both Ti types. At low pH, acceleration of ions exchange between Ti and saliva, and reduction of resistance of Ti surface against corrosion were observed (P < 0.05). Corrosion rate was also significantly increased in acidic medium (P < 0.05). Similar corrosion behavior was observed for both Ti types. The white-light-interferometry images of Ti surfaces show higher surface changes at low pH level. SEM images do not show detectable changes. No pitting corrosion was observed for any group. The pH level of artificial saliva influences the corrosion behavior of cp-Ti and Ti-6Al-4V alloy in that lower pH accelerates the corrosion rate and kinetics. The corrosion products may mitigate the survival rate of dental implants. © 2011 John Wiley & Sons A/S.

Microscopy and microanalysis 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bailey, G.W.; Corbett, J.M.; Dimlich, R.V.W.

1996-12-31

The Proceedings of this Annual Meeting contain paper of members from the three societies. These proceedings emphasizes the common research interests and attempts to eliminate some unwanted overlap. Topics covered are: microscopic analysis of animals with altered gene expression and in-situ gene and antibody localizations, high-resolution elemental mapping of nucleoprofein interactions, plant biology and pathology, quantitative HREM analysis of perfect and defected materials, computational methods for TEM image analysis, high-resolution FESM in materials research, frontiers in polymer microscopy and microanalysis, oxidation and corrosion, micro XRD and XRF, molecular microspectroscopy and spectral imaging, advances in confocal and multidimensional light microscopy, analyticalmore » electron microscopy in biology, correlative microscopy in biological sciences, grain-boundary microengineering, surfaces and interfaces, telepresence microscopy in education and research, MSA educational outreach, quantitative electron probe microanalysis, frontiers of analytical electron microscopy, critical issues in ceramic microstructures, dynamic organization of the cell, pathology, microbiology, high-resolution biological and cryo SEM, and scanning-probe microscopy.« less

Evaluation of the malaria rapid diagnostic test VIKIA malaria Ag Pf/Pan™ in endemic and non-endemic settings

PubMed Central

2013-01-01

Background Malaria rapid diagnostic tests (RDTs) are a useful tool in endemic malaria countries, where light microscopy is not feasible. In non-endemic countries they can be used as complementary tests to provide timely results in case of microscopy inexperience. This study aims to compare the new VIKIA Malaria Ag Pf/Pan™ RDT with PCR-corrected microscopy results and the commonly used CareStart™ RDT to diagnose falciparum and non-falciparum malaria in the endemic setting of Bamako, Mali and the non-endemic setting of Lyon, France. Methods Blood samples were collected during a 12-months and six-months period in 2011 from patients suspected to have malaria in Lyon and Bamako respectively. The samples were examined by light microscopy, the VIKIA Malaria Ag Pf/Pan™ test and in Bamako additionally with the CareStart™ RDT. Discordant results were corrected by real-time PCR. Sensitivity, specificity, positive predictive value and negative predictive value were used to evaluate test performance. Results Samples of 877 patients from both sites were included. The VIKIA Malaria Ag Pf/Pan™ had a sensitivity of 98% and 96% for Plasmodium falciparum in Lyon and Bamako, respectively, performing similar to PCR-corrected microscopy. Conclusions The VIKIA Malaria Ag Pf/Pan™ performs similar to PCR-corrected microscopy for the detection of P. falciparum, making it a valuable tool in malaria endemic and non-endemic regions. PMID:23742633

Evaluation of the malaria rapid diagnostic test VIKIA malaria Ag Pf/Pan™ in endemic and non-endemic settings.

PubMed

Eibach, Daniel; Traore, Boubacar; Bouchrik, Mourad; Coulibaly, Boubacar; Coulibaly, Nianégué; Siby, Fanta; Bonnot, Guillaume; Bienvenu, Anne-Lise; Picot, Stéphane

2013-06-06

Malaria rapid diagnostic tests (RDTs) are a useful tool in endemic malaria countries, where light microscopy is not feasible. In non-endemic countries they can be used as complementary tests to provide timely results in case of microscopy inexperience. This study aims to compare the new VIKIA Malaria Ag Pf/Pan™ RDT with PCR-corrected microscopy results and the commonly used CareStart™ RDT to diagnose falciparum and non-falciparum malaria in the endemic setting of Bamako, Mali and the non-endemic setting of Lyon, France. Blood samples were collected during a 12-months and six-months period in 2011 from patients suspected to have malaria in Lyon and Bamako respectively. The samples were examined by light microscopy, the VIKIA Malaria Ag Pf/Pan™ test and in Bamako additionally with the CareStart™ RDT. Discordant results were corrected by real-time PCR. Sensitivity, specificity, positive predictive value and negative predictive value were used to evaluate test performance. Samples of 877 patients from both sites were included. The VIKIA Malaria Ag Pf/Pan™ had a sensitivity of 98% and 96% for Plasmodium falciparum in Lyon and Bamako, respectively, performing similar to PCR-corrected microscopy. The VIKIA Malaria Ag Pf/Pan™ performs similar to PCR-corrected microscopy for the detection of P. falciparum, making it a valuable tool in malaria endemic and non-endemic regions.

Photoisomerisation and light-induced morphological switching of a polyoxometalate-azobenzene hybrid.

PubMed

Markiewicz, Grzegorz; Pakulski, Dawid; Galanti, Agostino; Patroniak, Violetta; Ciesielski, Artur; Stefankiewicz, Artur R; Samorì, Paolo

2017-06-29

The functionalization of a spherical Keplerate-type polyoxometalate {Mo 72 V 30 } with a cationic azobenzene surfactant has been achieved through ionic self-assembly. The photoisomerisation reaction of this complex, which emerges in a light-triggered aggregation-disaggregation process, has been followed by 1 H NMR spectroscopy, dynamic light scattering, absorption spectroscopy and scanning electron microscopy analyses.

Confocal microscopy as a useful approach to describe gill rakers of Asian species of carp and native filter-feeding fishes of the upper Mississippi River system

USGS Publications Warehouse

Liza R. Walleser,; D.R. Howard,; Sandheinrich, Mark B.; Gaikowski, Mark P.; Amberg, Jon J.

2014-01-01

To better understand potential diet overlap among exotic Asian species of carp and native species of filter-feeding fishes of the upper Mississippi River system, microscopy was used to document morphological differences in the gill rakers. Analysing samples first with light microscopy and subsequently with confocal microscopy, the three-dimensional structure of gill rakers in Hypophthalmichthys molitrix,Hypophthalmichthys nobilis and Dorosoma cepedianum was more thoroughly described and illustrated than previous work with traditional microscopy techniques. The three-dimensional structure of gill rakers in Ictiobus cyprinellus was described and illustrated for the first time.