The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis.

PubMed

Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong

2014-09-01

Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

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.

Open-source do-it-yourself multi-color fluorescence smartphone microscopy

PubMed Central

Sung, Yulung; Campa, Fernando; Shih, Wei-Chuan

2017-01-01

Fluorescence microscopy is an important technique for cellular and microbiological investigations. Translating this technique onto a smartphone can enable particularly powerful applications such as on-site analysis, on-demand monitoring, and point-of-care diagnostics. Current fluorescence smartphone microscope setups require precise illumination and imaging alignment which altogether limit its broad adoption. We report a multi-color fluorescence smartphone microscope with a single contact lens-like add-on lens and slide-launched total-internal-reflection guided illumination for three common tasks in investigative fluorescence microscopy: autofluorescence, fluorescent stains, and immunofluorescence. The open-source, simple and cost-effective design has the potential for do-it-yourself fluorescence smartphone microscopy. PMID:29188104

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

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

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.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

PubMed

Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

PubMed

Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

2013-07-29

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv. oryzae in planta

PubMed Central

Han, Sang-Wook; Park, Chang-Jin; Lee, Sang-Won; Ronald, Pamela C

2008-01-01

Background Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight disease, is a serious pathogen of rice. Here we describe a fluorescent marker system to study virulence and pathogenicity of X. oryzae pv. oryzae. Results A fluorescent X. oryzae pv. oryzae Philippine race 6 strain expressing green fluorescent protein (GFP) (PXO99GFP) was generated using the gfp gene under the control of the neomycin promoter in the vector, pPneo-gfp. The PXO99GFPstrain displayed identical virulence and avirulence properties as the wild type control strain, PXO99. Using fluorescent microscopy, bacterial multiplication and colonization were directly observed in rice xylem vessels. Accurate and rapid determination of bacterial growth was assessed using fluoremetry and an Enzyme-Linked ImmunoSorbant Assay (ELISA). Conclusion Our results indicate that the fluorescent marker system is useful for assessing bacterial infection and monitoring bacterial multiplication in planta. PMID:18826644

Investigation of autofocus algorithms for brightfield microscopy of unstained cells

NASA Astrophysics Data System (ADS)

Wu, Shu Yu; Dugan, Nazim; Hennelly, Bryan M.

2014-05-01

In the past decade there has been significant interest in image processing for brightfield cell microscopy. Much of the previous research on image processing for microscopy has focused on fluorescence microscopy, including cell counting, cell tracking, cell segmentation and autofocusing. Fluorescence microscopy provides functional image information that involves the use of labels in the form of chemical stains or dyes. For some applications, where the biochemical integrity of the cell is required to remain unchanged so that sensitive chemical testing can later be applied, it is necessary to avoid staining. For this reason the challenge of processing images of unstained cells has become a topic of increasing attention. These cells are often effectively transparent and appear to have a homogenous intensity profile when they are in focus. Bright field microscopy is the most universally available and most widely used form of optical microscopy and for this reason we are interested in investigating image processing of unstained cells recorded using a standard bright field microscope. In this paper we investigate the application of a range of different autofocus metrics applied to unstained bladder cancer cell lines using a standard inverted bright field microscope with microscope objectives that have high magnification and numerical aperture. We present a number of conclusions on the optimum metrics and the manner in which they should be applied for this application.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

PubMed

Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

2016-01-01

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Fluorescence (Multiwave) Confocal Microscopy.

PubMed

Welzel, J; Kästle, Raphaela; Sattler, Elke C

2016-10-01

In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

Experimental assessment of fluorescence microscopy signal enhancement by stimulated emission

NASA Astrophysics Data System (ADS)

Dake, Fumihiro; Yazawa, Hiroki

2017-10-01

The quantity of photons generated during fluorescence microscopy is principally determined by the quantum yield of the fluorescence dyes and the optical power of the excitation beam. However, even though low quantum yields can produce poor images, it is challenging to tune this parameter, while increasing the power of the excitation beam often results in photodamage. Here, we propose the use of stimulated emission (SE) as a means of enhancing both the signal intensity and signal-to-noise ratio during confocal fluorescence microscopy. This work experimentally confirmed that both these factors can be enhanced by SE radiation, through generating a greater number of photons than are associated with the standard fluorescence signal. We also propose the concept of stimulated emission enhancing fluorescence (SEEF) microscopy, which employs both the SE and fluorescence signals, and demonstrate that the intensity of an SEEF signal is greater than those of the individual SE and fluorescence signals.

Wide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations

NASA Astrophysics Data System (ADS)

Hofer, Matthias; Soeller, Christian; Brasselet, Sophie; Bertolotti, Jacopo

2018-04-01

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas.

PubMed

Sanai, Nader; Snyder, Laura A; Honea, Norissa J; Coons, Stephen W; Eschbacher, Jennifer M; Smith, Kris A; Spetzler, Robert F

2011-10-01

Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Fluorescence Microscopy of Single Molecules

ERIC Educational Resources Information Center

Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

2004-01-01

The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

Identification of powdered Chinese herbal medicines by fluorescence microscopy, Part 1: Fluorescent characteristics of mechanical tissues, conducting tissues, and ergastic substances.

PubMed

Wang, Ya-Qiong; Liang, Zhi-Tao; Li, Qin; Yang, Hua; Chen, Hu-Biao; Zhao, Zhong-Zhen; Li, Ping

2011-03-01

The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known. Copyright © 2010 Wiley-Liss, Inc.

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.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Fluorescence microscopy for the characterization of structural integrity

NASA Technical Reports Server (NTRS)

Street, Kenneth W.; Leonhardt, Todd A.

1991-01-01

The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

Fluorescence confocal microscopy for pathologists.

PubMed

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists' viewpoint.

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

PubMed Central

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-01-01

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327

Handheld Fluorescence Microscopy based Flow Analyzer.

PubMed

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

PubMed Central

Li, Yinghong; Yang, Yang; Guan, Xiangming

2012-01-01

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research. PMID:22794193

A series of fluorene-based two-photon absorbing molecules: synthesis, linear and nonlinear characterization, and bioimaging

PubMed Central

Andrade, Carolina D.; Yanez, Ciceron O.; Rodriguez, Luis; Belfield, Kevin D.

2010-01-01

The synthesis, structural, and photophysical characterization of a series of new fluorescent donor–acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescent quantum yields, and, significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy (2PFM) imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy. PMID:20481596

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

PubMed

Schaudinn, C; Carr, G; Gorur, A; Jaramillo, D; Costerton, J W; Webster, P

2009-08-01

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

Fluorescence microscopy: A tool to study autophagy

NASA Astrophysics Data System (ADS)

Rai, Shashank; Manjithaya, Ravi

2015-08-01

Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

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

Hwang, Hyun Sook; Kim, Soung Soo

Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATPmore » pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 deg C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.« less

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

NASA Astrophysics Data System (ADS)

Horton, Nicholas G.; Wang, Ke; Kobat, Demirhan; Clark, Catharine G.; Wise, Frank W.; Schaffer, Chris B.; Xu, Chris

2013-03-01

Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays.

PubMed

Zhang, Shaojuan

2016-01-01

Fluorescent probes are widely utilized for noninvasive fluorescence imaging. Continuing efforts have been made in developing novel fluorescent probes with improved fluorescence quantum yield, enhanced target-specificity, and lower cytotoxicity. Before such probes are administrated into a living system, it is essential to evaluate the subcellular uptake, targeting specificity, and cytotoxicity in vitro. In this chapter, we briefly outline common methods used to evaluate fluorescent probes using fluorescence microscopy, multiplate reader, and cytotoxicity assay.

A Comparative Study of Microscopic Images Captured by a Box Type Digital Camera Versus a Standard Microscopic Photography Camera Unit

PubMed Central

Desai, Nandini J.; Gupta, B. D.; Patel, Pratik Narendrabhai

2014-01-01

Introduction: Obtaining images of slides viewed by a microscope can be invaluable for both diagnosis and teaching.They can be transferred among technologically-advanced hospitals for further consultation and evaluation. But a standard microscopic photography camera unit (MPCU)(MIPS-Microscopic Image projection System) is costly and not available in resource poor settings. The aim of our endeavour was to find a comparable and cheaper alternative method for photomicrography. Materials and Methods: We used a NIKON Coolpix S6150 camera (box type digital camera) with Olympus CH20i microscope and a fluorescent microscope for the purpose of this study. Results: We got comparable results for capturing images of light microscopy, but the results were not as satisfactory for fluorescent microscopy. Conclusion: A box type digital camera is a comparable, less expensive and convenient alternative to microscopic photography camera unit. PMID:25478350

Anomalous doping of a molecular crystal monitored with confocal fluorescence microscopy: Terrylene in a p-terphenyl crystal

NASA Astrophysics Data System (ADS)

Białkowska, Magda; Deperasińska, Irena; Makarewicz, Artur; Kozankiewicz, Bolesław

2017-09-01

Highly terrylene doped single crystals of p-terphenyl, obtained by co-sublimation of both components, showed bright spots in the confocal fluorescence images. Polarization of the fluorescence excitation spectra, blinking and bleaching, and saturation behavior allowed us to attribute them to single molecules of terrylene anomalously embedded between two neighbor layers of the host crystal, in the (a,b) plane. Such an orientation of terrylene molecules results in much more efficient absorption and collection of the fluorescence photons than in the case of previously investigated molecules embedded in the substitution sites. The above conclusion was supported by quantum chemistry calculations. We postulate that the kind of doping considered in this work should be possible in other molecular crystals where the host molecules are organized in a herringbone pattern.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

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

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE PAGES

Brown, Koshonna; Thurn, Ted; Xin, Lun; ...

2017-07-19

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy

ERIC Educational Resources Information Center

Ito, Takashi

2008-01-01

This article describes experiments for an undergraduate instrumental analysis laboratory that aim to observe individual double-stranded DNA (dsDNA) molecules using fluorescence microscopy and atomic force microscopy (AFM). dsDNA molecules are observed under several different conditions to discuss their chemical and physical properties. In…

Temporal focusing microscopy combined with three-dimensional structured illumination

NASA Astrophysics Data System (ADS)

Isobe, Keisuke; Toda, Keisuke; Song, Qiyuan; Kannari, Fumihiko; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2017-05-01

Temporal focusing microscopy provides the optical sectioning capability in wide-field two-photon fluorescence imaging. Here, we demonstrate temporal focusing microscopy combined with three-dimensional structured illumination, which enables us to enhance the three-dimensional spatial resolution and reject the background fluorescence. Experimentally, the periodic pattern of the illumination was produced not only in the lateral direction but also in the axial direction by the interference between three temporal focusing pulses, which were easily generated using a digital micromirror device. The lateral resolution and optical sectioning capability were successfully enhanced by factors of 1.6 and 3.6, respectively, compared with those of temporal focusing microscopy. In the two-photon fluorescence imaging of a tissue-like phantom, the out-of-focus background fluorescence and the scattered background fluorescence could also be rejected.

Longitudinal in vivo two-photon fluorescence imaging

PubMed Central

Crowe, Sarah E.; Ellis-Davies, Graham C.R.

2014-01-01

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Lahn, Mattes; Hille, Carsten; Koberling, Felix; Kapusta, Peter; Dosche, Carsten

2010-02-01

Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

Saturated virtual fluorescence emission difference microscopy based on detector array

NASA Astrophysics Data System (ADS)

Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Ge, Baoliang; Wang, Wensheng; Liu, Xu

2017-07-01

Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

Autofluorescence of bovine ligamentum nuchae, cartilage, heart valve and lung measured by microscopy and fibre optics.

PubMed

Swatland, H J

1988-09-01

The fluorescence of bovine tissues was measured post mortem by microscopy of frozen sections and by using optical fibres to excite fluorescence and to measure fluorescence emission spectra. Mechanical disruption of the tissue (by comminution or sectioning) did not appreciably change tissue fluorescence spectra. Ligamentum nuchae had the strongest fluorescence and lung tissue had the weakest. In samples measured with a minimum prior exposure to ultraviolet light, the peak fluorescence emission was at 410 or 420 nm (with excitation at 365 nm). Exposure to ultraviolet light for about 1 minute shifted the fluorescence peak to 450 to 470 nm. Further exposure (about 30 minutes) caused a loss of the 450 to 470 nm fluorescence peak, while emissions above 530 nm were maintained or strengthened. Microscopy showed that the fluorescence that was measured by fibre optics from intact connective tissues originated mostly from collagen and elastin fibres.

Super-resolution fluorescence microscopy by stepwise optical saturation

PubMed Central

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

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.

Time-lapse microscopy of lung endothelial cells under hypoxia

NASA Astrophysics Data System (ADS)

Mehrvar, Shima; Ghanian, Zahra; Kondouri, Ganesh; Camara, Amadou S.; Ranji, Mahsa

2017-02-01

Objective: This study utilizes fluorescence microscopy to assess the effect of the oxygen tension on the production of reactive oxygen species (ROS) in mitochondria of fetal pulmonary artery endothelial cells (FPAECs). Introduction: Hypoxia is a severe oxygen stress, which mostly causes irreversible injury in lung cells. However, in some studies, it is reported that hypoxia decreases the severity of injuries. In this study, ROS production level was examined in hypoxic FPAECs treated with pentachlorophenol (PCP, uncoupler). This work was accomplished by monitoring and quantifying the changes in the level of the produced ROS in hypoxic cells before and after PCP treatment. Materials and methods: The dynamic of the mitochondrial ROS production in two groups of FPAECs was measured over time using time-lapse microscopy. For the first group, cells were incubated in 3% hypoxic condition for 2 hours and then continuously were exposed to hypoxic condition for imaging as well. For the second group, cells were incubated in normal oxygen condition. Time lapse images of the cells loaded with Mito-SOX (ROS indicator) were acquired, and the red fluorescence intensity profile of the cells was calculated. Changes in the level of the fluorescence intensity profile while they are treated with PCP indicates the dynamics of the ROS level. Results: The intensity profiles of the PCP-treated cells in the first group showed 47% lower ROS production rate than the PCP-treated cells in the second group. Conclusion: Time lapse microscopy revealed that hypoxic cells have lower ROS generation while treated with PCP. Therefore, this result suggests that hypoxia decreased electron transport chain activity in uncoupled chain.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Adaptive optical fluorescence microscopy.

PubMed

Ji, Na

2017-03-31

The past quarter century has witnessed rapid developments of fluorescence microscopy techniques that enable structural and functional imaging of biological specimens at unprecedented depth and resolution. The performance of these methods in multicellular organisms, however, is degraded by sample-induced optical aberrations. Here I review recent work on incorporating adaptive optics, a technology originally applied in astronomical telescopes to combat atmospheric aberrations, to improve image quality of fluorescence microscopy for biological imaging.

FluoroSim: A Visual Problem-Solving Environment for Fluorescence Microscopy

PubMed Central

Quammen, Cory W.; Richardson, Alvin C.; Haase, Julian; Harrison, Benjamin D.; Taylor, Russell M.; Bloom, Kerry S.

2010-01-01

Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data. FluoroSim renders synthetic fluorescence images from arbitrary geometric models represented as triangle meshes. We describe three rendering algorithms on graphics processing units for computing the convolution of the specimen model with a microscope's point-spread function and report on their performance. We also discuss several cases where the microscope simulator has been used to solve real problems in biology. PMID:20431698

Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques.

PubMed

Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

2017-01-01

We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

Advanced Methods in Fluorescence Microscopy

PubMed Central

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres. PMID:23271142

Advanced methods in fluorescence microscopy.

PubMed

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbe limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Advanced methods in fluorescence microscopy.

PubMed

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Nanodiamonds as multi-purpose labels for microscopy.

PubMed

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Enumerating viruses by using fluorescence and the nature of the nonviral background fraction.

PubMed

Pollard, Peter C

2012-09-01

Bulk fluorescence measurements could be a faster and cheaper way of enumerating viruses than epifluorescence microscopy, flow cytometry, or transmission electron microscopy (TEM). However, since viruses are not imaged, the background fluorescence compromises the signal, and we know little about its nature. In this paper the size ranges of nucleotides that fluoresce in the presence of SYBR gold were determined for wastewater and a range of freshwater samples using a differential filtration method. Fluorescence excitation-emission matrices (FEEMs) showed that >70% of the SYBR fluorescence was in the <10-nm size fraction (background) and was not associated with intact viruses. This was confirmed using TEM. The use of FEEMs to develop a fluorescence-based method for counting viruses is an approach that is fundamentally different from the epifluorescence microscopy technique used for enumerating viruses. This high fluorescence background is currently overlooked, yet it has had a most pervasive influence on the development of a simple fluorescence-based method for quantifying viral abundance in water.

Association of Candida sp. with the Degrees of Dysplasia and Oral Cancer: A Study by Calcofluor White under Fluorescent Microscopy

PubMed Central

Tamgadge, Sandhya; Tamgadge, Avinash; Pillai, Aswathy; chande, Mayura; Acharya, Siddharth; Kamat, Narayan

2017-01-01

Background and objective: Candida albicans (C. albicans) play a significant role in oral mucosal carcinogenesis. It can be identified using various techniques in cytological smears. But, very few studies have been conducted on histopathological sections using calcofluor white M2R under fluorescent microscopy. Additionally, detection and quantification of Candida colonies and its correlation with various grades of oral leukoplakia and oral carcinomas have not been explored much. Methods: The current retrospective study included 80 samples from archives consisting of 60 samples in the study group (10 cases each of mild, moderate, and severe epithelial dysplasia (totally 30) and 30 cases of oral carcinoma). Sections were stained with calcofluor white (CFW) and 10% KOH for the observation under fluorescent microscopy and correlated with different grades of oral leukoplakia and oral carcinomas. Chi-square test was used in SSPS software to study the presence and absence of Candida sp. in different groups. Results: The study groups of oral carcinoma and dysplasia showed a significant association with Candida sp. (P=0). When carcinoma was compared with each grade of dysplasia, except mild dysplasia (P=4.4E-05), both moderate (P=0.402195) and severe dysplasia (P=0.558746) showed an insignificant P-value. When the groups of mild (13.3%), moderate (30%), and severe (33.3%) dysplasia were considered independently, the incidence of Candida sp. increased as the grade of dysplasia increased. The number of colonies have been counted and the maximum number of colonies have been observed in carcinoma and the least have been observed in mild dysplasia. Conclusion: A significant association of Candida colonies with epithelial dysplasia and oral cancer was established. Further, CFW was found a promising candidate to identify Candida colonies in tissue sections using fluorescent microscopy. PMID:29563930

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

PubMed

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells

PubMed Central

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W.; Gautier, Virginie W.

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip. PMID:26485569

Widefield fluorescence sectioning with HiLo microscopy.

PubMed

Mertz, Jerome; Lim, Daryl; Chu, Kengyeh K; Bozinovic, Nenad; Ford, Timothy

2009-01-01

HiLo microscopy is a widefield fluorescence imaging technique that provides depth discrimination by combining two images, one with non-uniform illumination and one with uniform illumination. We discuss the theory of this technique and a variety of practical implementations in brain-tissue imaging and fluorescence endomicroscopy.

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

Miranda, Adelaide; De Beule, Pieter A. A., E-mail: pieter.de-beule@inl.int; Martins, Marco

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discussmore » sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.« less

Tracking Lithium Ions via Widefield Fluorescence Microscopy for Battery Diagnostics.

PubMed

Padilla, Nicolas A; Rea, Morgan T; Foy, Michael; Upadhyay, Sunil P; Desrochers, Kyle A; Derus, Tyler; Knapper, Kassandra A; Hunter, Nathanael H; Wood, Sharla; Hinton, Daniel A; Cavell, Andrew C; Masias, Alvaro G; Goldsmith, Randall H

2017-07-28

Direct tracking of lithium ions with time and spatial resolution can provide an important diagnostic tool for understanding mechanisms in lithium ion batteries. A fluorescent indicator of lithium ions, 2-(2-hydroxyphenyl)naphthoxazole, was synthesized and used for real-time tracking of lithium ions via widefield fluorescence microscopy. The fluorophore can be excited with visible light and was shown to enable quantitative determination of the lithium ion diffusion constant in a microfluidic model system for a plasticized polymer electrolyte lithium battery. The use of widefield fluorescence microscopy for in situ tracking of lithium ions in batteries is discussed.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Probing Membrane Order and Topography in Supported Lipid Bilayers by Combined Polarized Total Internal Reflection Fluorescence-Atomic Force Microscopy

PubMed Central

Oreopoulos, John; Yip, Christopher M.

2009-01-01

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C20 or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions. PMID:19254557

Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2

NASA Astrophysics Data System (ADS)

McConnell, Gail; Riis, Erling

2004-10-01

We report on a novel and compact reliable laser source capable of short-wavelength two-photon laser scanning fluorescence microscopy based on soliton self-frequency shift effects in photonic crystal fibre. We demonstrate the function of the system by performing two-photon microscopy of smooth muscle cells and cardiac myocytes from the rat pulmonary vein and Chinese hamster ovary cells loaded with the fluorescent calcium indicator fura-2/AM.

Advanced fluorescence microscopy techniques for the life sciences

PubMed Central

Aguib, Yasmine; Yacoub, Magdi H.

The development of super-resolved fluorescence microscopy, for which the Nobel Prize was awarded in 2014, has been a topic of interest to physicists and biologists alike. It is inevitable that numerous questions in biomedical research cannot be answered by means other than direct observation. In this review, advances to fluorescence microscopy are covered in a widely accessible fashion to facilitate its use in decisions related to its acquisition and utilization in biomedical research. PMID:29043264

DNA origami-based standards for quantitative fluorescence microscopy.

PubMed

Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

2014-01-01

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE PAGES

Ducic, Tanja; Paunesku, Tatjana; Chen, Si; ...

2016-12-09

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

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

Ducic, Tanja; Paunesku, Tatjana; Chen, Si

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

Detection of SiO2 nanoparticles in lung tissue by ToF-SIMS imaging and fluorescence microscopy.

PubMed

Veith, Lothar; Vennemann, Antje; Breitenstein, Daniel; Engelhard, Carsten; Wiemann, Martin; Hagenhoff, Birgit

2017-07-10

The direct detection of nanoparticles in tissues at high spatial resolution is a current goal in nanotoxicology. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is widely used for the direct detection of inorganic and organic substances with high spatial resolution but its capability to detect nanoparticles in tissue sections is still insufficiently explored. To estimate the applicability of this technique for nanotoxicological questions, comparative studies with established techniques on the detection of nanoparticles can offer additional insights. Here, we compare ToF-SIMS imaging data with sub-micrometer spatial resolution to fluorescence microscopy imaging data to explore the usefulness of ToF-SIMS for the detection of nanoparticles in tissues. SiO 2 nanoparticles with a mean diameter of 25 nm, core-labelled with fluorescein isothiocyanate, were intratracheally instilled into rat lungs. Subsequently, imaging of lung cryosections was performed with ToF-SIMS and fluorescence microscopy. Nanoparticles were successfully detected with ToF-SIMS in 3D microanalysis mode based on the lateral distribution of SiO 3 - (m/z 75.96), which was co-localized with the distribution pattern that was obtained from nanoparticle fluorescence. In addition, the lateral distribution of protein (CN - , m/z 26.00) and phosphate based signals (PO 3 - , m/z 78.96) originating from the tissue material could be related to the SiO 3 - lateral distribution. In conclusion, ToF-SIMS is suitable to directly detect and laterally resolve SiO 2 nanomaterials in biological tissue at sufficient intensity levels. At the same time, information about the chemical environment of the nanoparticles in the lung tissue sections is obtained.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

PubMed

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

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.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

PubMed Central

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-01-01

Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

DOE PAGES

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry; ...

2016-08-31

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

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

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy

PubMed Central

Liv, Nalan; van Oosten Slingeland, Daan S. B.; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W.; Hoogenboom, Jacob P.

2016-01-01

We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization. PMID:26580231

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

PubMed

Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

2013-08-09

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Projected Uses of Cellular Models and Fluorescence Microscopy for Identification of Antivesicants

DTIC Science & Technology

1993-05-13

AD-P008 761 PROJECTED USES OF CELLULAR MODELS AND FLUORESCENCE MICROSCOPY FOR IDENTIFICATION OF ANTIVESICANTS Millard M. Mershon, Stacey M...epidermal keratinocytes (NHEK), fluorescent dye marker probes and spectrofluorometry led to a preliminary feasibility study’ This showed that the...acetoxymethyl ester that is taken into cells and cleaved by intracellular esterases’. It remains as a fluorescent marker until it leaks out through damaged

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

NASA Astrophysics Data System (ADS)

Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

2014-02-01

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.

PubMed

Burnette, Dylan T; Sengupta, Prabuddha; Dai, Yuhai; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2011-12-27

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

The X-ray Fluorescence Microscopy Beamline at the Australian Synchrotron

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

Paterson, D.; Jonge, M. D. de; Howard, D. L.

2011-09-09

A hard x-ray micro-nanoprobe has commenced operation at the Australian Synchrotron providing versatile x-ray fluorescence microscopy across an incident energy range from 4 to 25 keV. Two x-ray probes are used to collect {mu}-XRF and {mu}-XANES for elemental and chemical microanalysis: a Kirkpatrick-Baez mirror microprobe for micron resolution studies and a Fresnel zone plate nanoprobe capable of 60-nm resolution. Some unique aspects of the beamline design and operation are discussed. An advanced energy dispersive x-ray fluorescence detection scheme named Maia has been developed for the beamline, which enables ultrafast x-ray fluorescence microscopy.

Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

NASA Astrophysics Data System (ADS)

Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

2011-03-01

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

NicoLase—An open-source diode laser combiner, fiber launch, and sequencing controller for fluorescence microscopy

PubMed Central

Walsh, James; Böcking, Till; Gaus, Katharina

2017-01-01

Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available. PMID:28301563

Dual-modality imaging with 99mTc and fluorescent indocyanine green using surface-modified silica nanoparticles for biopsy of the sentinel lymph node: an animal study

PubMed Central

2013-01-01

Background We propose a new approach to facilitate sentinel node biopsy examination by multimodality imaging in which radioactive and near-infrared (NIR) fluorescent nanoparticles depict deeply situated sentinel nodes and fluorescent nodes with anatomical resolution in the surgical field. For this purpose, we developed polyamidoamine (PAMAM)-coated silica nanoparticles loaded with technetium-99m (99mTc) and indocyanine green (ICG). Methods We conducted animal studies to test the feasibility and utility of this dual-modality imaging probe. The mean diameter of the PAMAM-coated silica nanoparticles was 30 to 50 nm, as evaluated from the images of transmission electron microscopy and scanning electron microscopy. The combined labeling with 99mTc and ICG was verified by thin-layer chromatography before each experiment. A volume of 0.1 ml of the nanoparticle solution (7.4 MBq, except for one rat that was injected with 3.7 MBq, and 1 μg of an ICG derivative [ICG-sulfo-OSu]) was injected submucosally into the tongue of six male Wistar rats. Results Scintigraphic images showed increased accumulation of 99mTc in the neck of four of the six rats. Nineteen lymph nodes were identified in the dissected neck of the six rats, and a contact radiographic study showed three nodes with a marked increase in uptake and three nodes with a weak uptake. NIR fluorescence imaging provided real-time clear fluorescent images of the lymph nodes in the neck with anatomical resolution. Six lymph nodes showed weak (+) to strong (+++) fluorescence, whereas other lymph nodes showed no fluorescence. Nodes showing increased radioactivity coincided with the fluorescent nodes. The radioactivity of 15 excised lymph nodes from the four rats was assayed using a gamma well counter. Comparisons of the levels of radioactivity revealed a large difference between the high-fluorescence-intensity group (four lymph nodes; mean, 0.109% ± 0.067%) and the low- or no-fluorescence-intensity group (11 lymph nodes; mean, 0.001% ± 0.000%, p < 0.05). Transmission electron microscopy revealed that small black granules were localized to and dispersed within the cytoplasm of macrophages in the lymph nodes. Conclusion Although further studies are needed to determine the appropriate dose of the dual-imaging nanoparticle probe for effective sensitivity and safety, the results of this animal study revealed a novel method for improved node detection by a dual-modality approach for sentinel lymph node biopsy. PMID:23618132

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

PubMed

Wang, Yilin; Kanchanawong, Pakorn

2016-12-01

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

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.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

PubMed Central

Siegel, Nisan; Brooker, Gary

2014-01-01

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

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

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.

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy.

PubMed

Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

2016-06-01

We recently proposed a straightforward fluorescence microscopy technique to study adhesion of Giant Unilamellar Vesicles. This technique is based on dual observations which combine epi-fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy: TIRF images are normalized by epi-fluorescence ones. By this way, it is possible to map the membrane/substrate separation distance with a nanometric resolution, typically ~20 nm, with a maximal working range of 300-400 nm. The purpose of this paper is to demonstrate that this technique is useful to quantify vesicle adhesion from ultra-weak to strong membrane-surface interactions. Thus, we have examined unspecific and specific adhesion conditions. Concerning unspecific adhesion, we have controlled the strength of electrostatic forces between negatively charged vesicles and various functionalized surfaces which exhibit a positive or a negative effective charge. Specific adhesion was highlighted with lock-and-key forces mediated by the well defined biotin/streptavidin recognition. Copyright © 2016 Elsevier B.V. All rights reserved.

Applications of two-photon fluorescence microscopy in deep-tissue imaging

NASA Astrophysics Data System (ADS)

Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

2000-07-01

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array

PubMed Central

Wu, Jianglai; Tang, Anson H. L.; Mok, Aaron T. Y.; Yan, Wenwei; Chan, Godfrey C. F.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2017-01-01

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged. PMID:28966855

Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing.

PubMed

Oommen, Seema; Banaji, Nandita

2017-01-01

There have been rapid technological advances in the detection of Mycobacterium tuberculosis and its drug susceptibility in clinical samples. These include advances in microscopic examination, in vitro culture and application of molecular techniques. The World Health Organization (WHO) has played a large role in evaluating these technologies for their efficacy and feasibility, especially in the developing countries. Amongst these, the Revised National Tuberculosis Control Programme (RNTCP), through its national network of designated microscopy centres and intermediate reference laboratories, has adopted certain technologies that are currently implemented in India. Advances in microscopy technology include fluorescent microscopy using light-emitting diode source, sodium hypochlorite microscopy and vital fluorescent staining of sputum smears. Automation of in vitro culture has markedly reduced the turnaround time (TAT), even in smear-negative samples, and permits simultaneous detection of resistant mutants. Molecular detection of drug resistance has further reduced the TAT, and the cartridge-based nucleic acid amplification test with its performance convenience and rapid results, appears poised to become the future of tuberculosis (TB) diagnosis in all settings, provided the cost of testing is reduced especially for use in private diagnostic settings. This article reviews technologies currently available for the diagnosis of TB, keeping in mind the WHO recommendations and the RNTCP practices. This is a thematic synthesis of data available on diagnosis in literature, preserving the conclusions of the primary studies.

Photocontrollable Fluorescent Proteins for Superresolution Imaging

PubMed Central

Shcherbakova, Daria M.; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V.

2014-01-01

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine. PMID:24895855

Single-molecule imaging of cytoplasmic dynein in vivo.

PubMed

Ananthanarayanan, Vaishnavi; Tolić, Iva M

2015-01-01

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior. Copyright © 2015 Elsevier Inc. All rights reserved.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Chlorophyll and its degradation products in the two-spotted spider mite, Tetranychus urticae: observations using epifluorescence and confocal laser scanning microscopy.

PubMed

Occhipinti, Andrea; Maffei, Massimo E

2013-10-01

Chlorophyll and chlorophyll degradation products were observed in the two-spotted spider mite (Tetranychus urticae) using epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM). A clear red fluorescence (EFM) and a fluorescence induced by a laser wavelength of 650 nm (CLSM) were observed. In the lateral caeca, in the ventriculus and in the excretory organ, a bright light blue fluorescence was observed in close association with chlorophyll by using EFM. The same material can be localized with CLSM by using a laser with a wavelength of 488 nm. By comparison with synthetic guanine, this bright fluorescence is supposed to be guanine. The presence of guanine fluorescence in the mite pellets confirms this hypothesis. A possible mechanism for guanine formation is discussed.

Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells.

PubMed

Day, Richard N; Davidson, Michael W

2012-05-01

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. Copyright © 2012 WILEY Periodicals, Inc.

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.

In Vivo Near-Infrared Imaging of Fibrin Deposition in Thromboembolic Stroke in Mice

PubMed Central

Zhang, Yi; Fan, Shufeng; Yao, Yuyu; Ding, Jie; Wang, Yu; Zhao, Zhen; Liao, Lei; Li, Peicheng; Zang, Fengchao; Teng, Gao-Jun

2012-01-01

Objectives Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging. Materials and Methods The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia. Results In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume. Conclusion Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke. PMID:22272319

Frequency-domain photoacoustic and fluorescence microscopy: application on labeled and unlabeled cells

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Pfeffer, Karoline; Wohlfarth, Sven; Hannesschläger, Günther; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.

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.

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

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.

Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

PubMed

Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

2014-05-01

The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

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

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

PubMed

Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

2015-09-01

The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real-time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue

PubMed Central

Egea, Isabel; Bian, Wanping; Barsan, Cristina; Jauneau, Alain; Pech, Jean-Claude; Latché, Alain; Li, Zhengguo; Chervin, Christian

2011-01-01

Background and Aims There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase. Methods Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices. Key results At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts. Conclusions These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes. PMID:21788376

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

PubMed

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Image scanning fluorescence emission difference microscopy based on a detector array.

PubMed

Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

2017-06-01

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

PubMed Central

Zhu, Hongying; Ozcan, Aydogan

2013-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

PubMed

Puchkov, Evgeny O

2010-06-01

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 microm size, stainable by a fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

Coherent Anti-Stokes Raman Scattering (CARS) Microscopy: A Novel Technique for Imaging the Retina

PubMed Central

Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Lei, Tim C.

2013-01-01

Purpose. To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Methods. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. Results. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. Conclusions. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice. PMID:23580484

Localization-based super-resolution imaging of cellular structures.

PubMed

Kanchanawong, Pakorn; Waterman, Clare M

2013-01-01

Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction to ~250 nm, prompting the development of super-resolution microscopy which offers resolution approaching the scale of single proteins, i.e., ~20 nm. Here, we describe protocols for single molecule localization-based super-resolution imaging, using focal adhesion proteins as an example and employing either photoswitchable fluorophores or photoactivatable fluorescent proteins. These protocols should also be easily adaptable to imaging a broad array of macromolecular assemblies in cells whose components can be fluorescently tagged and assemble into high density structures.

Applying fluorescence microscopy to the investigation of the behavior of foodborne pathogens on produce

NASA Astrophysics Data System (ADS)

Brandl, Maria T.

2009-05-01

In the past decade, the development of new tools to better visualize microbes at the cellular scale has spurred a renaissance in the application of microscopy to the study of bacteria in their natural environment. This renewed interest in microscopy may be largely attributable to the advent of the confocal laser scanning microscope (CLSM) and to the discovery of the green fluorescent protein. This article provides information about the use of fluorescence microscopy combined with fluorescent labels such as GFP, DsRed, and DNA stains, with immunofluorescence, and with digital image analysis, to examine the behavior of bacteria and other microbes on plant surfaces. Some of the advantages and pitfalls of these methods will be described using practical examples derived from studies of the ecology of foodborne pathogens, namely Salmonella enterica and E. coli O157:H7, on fresh fruit and vegetables. Confocal microscopy has been a powerful approach to uncover some of the factors involved in the association of produce with epidemics caused by these human pathogens and their interaction with other microbes in their nonhost environment.

Characterization of human carotid atherosclerotic tissues imaged by combining multiple multiphoton microscopy techniques

NASA Astrophysics Data System (ADS)

Baria, E.; Cicchi, R.; Nesi, G.; Massi, D.; Pavone, F. S.

2017-07-01

We combined Second Harmonic Generation, Two-Photon Fluorescence and Fluorescence Lifetime Imaging Microscopy for studying human carotid ex vivo tissue sections affected by atherosclerosis, resulting in the discrimination of different arterial regions within the plaques.

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.

Analysis of cholesterol trafficking with fluorescent probes

PubMed Central

Maxfield, Frederick R.; Wüstner, Daniel

2013-01-01

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly. PMID:22325611

3D fluorescence anisotropy imaging using selective plane illumination microscopy.

PubMed

Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

2015-08-24

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Near-Infrared Fluorescence Imaging of Liver Metastases in Rats using Indocyanine Green

PubMed Central

van der Vorst, Joost R.; Hutteman, Merlijn; Mieog, Sven D.; de Rooij, Karien E.; Kaijzel, Eric L.; Löwik, Clemens W.G.M.; Putter, Hein; Kuppen, Peter J.K.; Frangioni, John V.; van de Velde, Cornelis J.H.; Vahrmeijer, Alexander L.

2011-01-01

Background Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is a promising technique to obtain real-time assessment of the extent and number of colorectal liver metastases during surgery. The current study aims to optimize dosage and timing of ICG administration. Materials and methods Liver tumors were induced in 18 male WAG/Rij rats by subcapsular inoculation of CC531 rat colorectal cancer cells into three distinct liver lobes. Rats were divided in 2 groups: imaging after 24 and 48 hours or 72 and 96 hours after intravenous ICG administration. In each time group, rats were allocated to three dose groups: 0.04, 0.08, or 0.16 mg ICG. Intraoperative imaging and ex vivo measurements were performed using Mini-FLARE™ and confirmed by fluorescence microscopy. Fluorescence intensity was quantified using the Mini-FLARE software and the difference between tumor signal and liver signal (tumor-to-liver ratio; TLR) was calculated. Results In all 18 rats, all colorectal liver metastases (N = 34), some as small as 1.2 mm, were identified using ICG and the Mini-FLARE™ imaging system. Average tumor-to-liver ratio (TLR) over all groups was 3.0 ± 1.2. TLR was significantly higher in the 72 h time group compared to other time points. ICG dose did not significantly influence TLR, but a trend was found favoring the 0.08 mg dose group. Fluorescence microscopy demonstrated a clear fluorescent rim around the tumor. Conclusions This study demonstrates that colorectal cancer liver metastases can be clearly identified during surgery using ICG and the Mini-FLARE™ imaging system, with optimal timing of 72 h post-injection and an optimal dose of 0.08 mg (0.25 mg/kg) ICG. NIR fluorescence imaging has the potential to improve intraoperative detection of micrometastases and thus the completeness of resection. PMID:21396660

Visualization of melanoma tumor with lectin-conjugated rare-earth doped fluoride nanocrystals

PubMed Central

Dumych, Tetiana; Lutsyk, Maxym; Banski, Mateusz; Yashchenko, Antonina; Sojka, Bartlomiej; Horbay, Rostyslav; Lutsyk, Alexander; Stoika, Rostyslav; Misiewicz, Jan; Podhorodecki, Artur; Bilyy, Rostyslav

2014-01-01

Aim To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC). Methods B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu3+-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu3+ with the fluorescent emission at 600-720 nm range. Results NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu3+ conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months. Conclusion NPL lectin-NaGdF4:Eu3+ conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor. PMID:24891277

Microscopy basics and the study of actin-actin-binding protein interactions.

PubMed

Thomasson, Maggie S; Macnaughtan, Megan A

2013-12-15

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Labeling TiO2 nanoparticles with dyes for optical fluorescence microscopy and determination of TiO2-DNA nanoconjugate stability.

PubMed

Thurn, Kenneth T; Paunesku, Tatjana; Wu, Aiguo; Brown, Eric M B; Lai, Barry; Vogt, Stefan; Maser, Jörg; Aslam, Mohammed; Dravid, Vinayak; Bergan, Raymond; Woloschak, Gayle E

2009-06-01

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO2) nanoparticles. These nanoparticles, when electronically linked to single-stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO2-DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO2 nanoparticles and TiO2-DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO2 nanoparticle uptake in a large population of living cells (>10(4) cells). X-ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO2-DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO2-DNA nanoconjugates in malignant cells.

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

NASA Astrophysics Data System (ADS)

Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation.

PubMed

Malkani, Naila; Schmid, Johannes A

2011-04-07

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins. When we applied a commonly used FRET microscopy technique--the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10-15% after illumination at the YFP-excitation wavelength--a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor. Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

PubMed Central

Knoblauch, Jan; Peters, Winfried S.; Knoblauch, Michael

2016-01-01

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks. PMID:26929203

Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

NASA Astrophysics Data System (ADS)

Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

2015-05-01

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1 immunolabeling. See DOI: 10.1039/c5nr01539a

Low-cost fluorescence microscopy for point-of-care cell imaging

NASA Astrophysics Data System (ADS)

Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

2010-02-01

Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins[S

PubMed Central

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

2016-01-01

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques. PMID:27565170

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.

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.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

PubMed

Baroux, Célia; Schubert, Veit

2018-01-01

In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.

PubMed

Yoshitake, Tadayuki; Giacomelli, Michael G; Cahill, Lucas C; Schmolze, Daniel B; Vardeh, Hilde; Faulkner-Jones, Beverly E; Connolly, James L; Fujimoto, James G

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

PubMed Central

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-01-01

Abstract. Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue. PMID:28032121

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

NASA Astrophysics Data System (ADS)

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization

PubMed Central

Schallek, Jesse; Geng, Ying; Nguyen, HoanVu; Williams, David R.

2013-01-01

Purpose. To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina. Methods. Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc. Results. We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm2 of retinal area). Conclusions. We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease. PMID:24150762

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

PubMed

Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

2013-12-01

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Techniques for super-resolution microscopy using NV-diamond

NASA Astrophysics Data System (ADS)

Trifonov, Alexei; Glenn, David; Bar-Gill, Nir; Le Sage, David; Walsworth, Ronald

2011-05-01

We discuss the development and application of techniques for super-resolution microscopy using NV centers in diamond: stimulated emission depletion (STED), metastable ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM). NV centers do not bleach under optical excitation, are not biotoxic, and have long-lived electronic spin coherence and spin-state-dependent fluorescence. Thus NV-diamond has great potential as a fluorescent biomarker and as a magnetic biosensor.

Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles

PubMed Central

Ichimura, Taro; Jin, Takashi; Fujita, Hideaki; Higuchi, Hideo; Watanabe, Tomonobu M.

2014-01-01

Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies. PMID:25120488

Segmentation and Morphometric Analysis of Cells from Fluorescence Microscopy Images of Cytoskeletons

PubMed Central

Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

2013-01-01

We developed a method to reconstruct cell geometry from confocal fluorescence microscopy images of the cytoskeleton. In the method, region growing was implemented twice. First, it was applied to the extracellular regions to differentiate them from intracellular noncytoskeletal regions, which both appear black in fluorescence microscopy imagery, and then to cell regions for cell identification. Analysis of morphological parameters revealed significant changes in cell shape associated with cytoskeleton disruption, which offered insight into the mechanical role of the cytoskeleton in maintaining cell shape. The proposed segmentation method is promising for investigations on cell morphological changes with respect to internal cytoskeletal structures. PMID:23762186

Segmentation and morphometric analysis of cells from fluorescence microscopy images of cytoskeletons.

PubMed

Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

2013-01-01

We developed a method to reconstruct cell geometry from confocal fluorescence microscopy images of the cytoskeleton. In the method, region growing was implemented twice. First, it was applied to the extracellular regions to differentiate them from intracellular noncytoskeletal regions, which both appear black in fluorescence microscopy imagery, and then to cell regions for cell identification. Analysis of morphological parameters revealed significant changes in cell shape associated with cytoskeleton disruption, which offered insight into the mechanical role of the cytoskeleton in maintaining cell shape. The proposed segmentation method is promising for investigations on cell morphological changes with respect to internal cytoskeletal structures.

Safranine fluorescent staining of wood cell walls.

PubMed

Bond, J; Donaldson, L; Hill, S; Hitchcock, K

2008-06-01

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies

PubMed Central

Sun, Ning; Shibata, Brad; Hess, John F.

2015-01-01

Purpose Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. Methods A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. Results An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence. Conclusions A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic. PMID:25991907

Three-dimensional image formation in fiber-optical second-harmonic-generation microscopy.

PubMed

Gu, Min; Fu, Ling

2006-02-06

Three-dimensional (3-D) image formation in fiber-optical second-harmonic-generation microscopy is revealed to be purely coherent and therefore can be described by a 3-D coherent transfer function (CTF) that exhibits the same spatial frequency passband as that of fiber-optical reflection-mode non-fluorescence microscopy. When the numerical aperture of the fiber is much larger than the angle of convergence of the illumination on the fiber aperture, the performance of fiber-optical second-harmonic-generation microscopy behaves as confocal second-harmonic-generation microscopy. The dependence of axial resolution on fiber coupling parameters shows an improvement of approximately 7%, compared with that in fiber-optical two-photon fluorescence microscopy.

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2016-10-01

lipopolysaccharide, capsular polysaccharide , and peptidoglycan simultaneously in live anaerobic commensal bacteria. This technology enabled us to track the...endotoxin, capsular polysaccharide , inflammatory bowel disease,microbiome microbiota, carbohydrate chemistry, fluorescent microscopy, 2-photon...lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory bowel disease, microbiome, microbiota, carbohydrate chemistry, fluorescent microscopy

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

PubMed Central

Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E.

2014-01-01

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression. PMID:25232010

Detection of polycyclic aromatic hydrocarbons (PAHs) in Medicago sativa L. by fluorescence microscopy.

PubMed

Alves, Wilber S; Manoel, Evelin A; Santos, Noemi S; Nunes, Rosane O; Domiciano, Giselli C; Soares, Marcia R

2017-04-01

Green technologies, such as phytoremediation, are effective for removing organic pollutants derived from oil and oil products, including polycyclic aromatic hydrocarbons (PAHs). Given the increasing popularity of these sustainable remediation techniques, methods based on fluorescence microscopy and multiphoton microscopy for the environmental monitoring of such pollutants have emerged in recent decades as effective tools for phytoremediation studies aimed at understanding the fate of these contaminants in plants. However, little is known about the cellular and molecular mechanisms involved in PAH uptake, responses and degradation by plants. Thus, the present study aimed to detect the location of pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then collected and observed under a fluorescence microscope to detect the presence of PAHs in various tissues. One important finding of the present study was intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with a previously unknown function, may be sites of PAH conjugation and degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

PubMed

Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles

2015-02-01

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

A Patch-Based Method for Repetitive and Transient Event Detection in Fluorescence Imaging

PubMed Central

Boulanger, Jérôme; Gidon, Alexandre; Kervran, Charles; Salamero, Jean

2010-01-01

Automatic detection and characterization of molecular behavior in large data sets obtained by fast imaging in advanced light microscopy become key issues to decipher the dynamic architectures and their coordination in the living cell. Automatic quantification of the number of sudden and transient events observed in fluorescence microscopy is discussed in this paper. We propose a calibrated method based on the comparison of image patches expected to distinguish sudden appearing/vanishing fluorescent spots from other motion behaviors such as lateral movements. We analyze the performances of two statistical control procedures and compare the proposed approach to a frame difference approach using the same controls on a benchmark of synthetic image sequences. We have then selected a molecular model related to membrane trafficking and considered real image sequences obtained in cells stably expressing an endocytic-recycling trans-membrane protein, the Langerin-YFP, for validation. With this model, we targeted the efficient detection of fast and transient local fluorescence concentration arising in image sequences from a data base provided by two different microscopy modalities, wide field (WF) video microscopy using maximum intensity projection along the axial direction and total internal reflection fluorescence microscopy. Finally, the proposed detection method is briefly used to statistically explore the effect of several perturbations on the rate of transient events detected on the pilot biological model. PMID:20976222

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

PubMed Central

Kopek, Benjamin G.; Shtengel, Gleb; Xu, C. Shan; Clayton, David A.; Hess, Harald F.

2012-01-01

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to “colorize” detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging. PMID:22474357

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

PubMed Central

Pratx, Guillem; Chen, Kai; Sun, Conroy; Martin, Lynn; Carpenter, Colin M.; Olcott, Peter D.; Xing, Lei

2012-01-01

Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers. PMID:23056276

Operational Implementation of LED Fluorescence Microscopy in Screening Tuberculosis Suspects in an Urban HIV Clinic in Uganda

PubMed Central

Albert, Heidi; Nakiyingi, Lydia; Sempa, Joseph; Mbabazi, Olive; Mukkada, Sheena; Nyesiga, Barnabas; Perkins, Mark D.; Manabe, Yukari C.

2013-01-01

Background Light emitting diode (LED) fluorescence microscopy (FM) is an affordable, technology targeted for use in resource-limited settings and recommended for widespread roll-out by the World Health Organization (WHO). We sought to compare the operational performance of three LED FM methods compared to light microscopy in a cohort of HIV-positive tuberculosis (TB) suspects at an urban clinic in a high TB burden country. Methods Two spot specimens collected from TB suspects were included in the study. Smears were stained using auramine O method and read after blinding by three LED-based FM methods by trained laboratory technicians in the Infectious Diseases Institutelaboratory. Leftover portions of the refrigerated sputum specimens were transported to the FIND Tuberculosis Research Laboratory for Ziehl Neelsen (ZN) smear preparation and reading by experienced technologist as well as liquid and solid culture. Results 174 of 627 (27.8%) specimens collected yielded one or more positive mycobacterial cultures. 94.3% (164/174) were M. tuberculosis complex. LED FM was between 7.3–11.0% more sensitive compared to ZN microscopy. Of the 592 specimens examined by all microscopy methods, there was no significant difference in sensitivity between the three LED FM methods. The specificity of the LED FM methods was between 6.1% and 7.7% lower than ZN microscopy (P<0.001), although exclusion of the single poor reader resulted in over 98% specificity for all FM methods. Conclusions Laboratory technicians in routine settings can be trained to use FM which is more sensitive than ZN microscopy. Despite rigorous proficiency testing, there were operator-dependent accuracy issues which highlight the critical need for intensive quality assurance procedures during LED FM implementation. The low sensitivity of FM for HIV-positive individuals particularly those with low CD4 T cell counts, will limit the number of additional patients found by LED FM in countries with high rates of HIV co-infection. PMID:24039780

Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

PubMed

Hezel, Marcus; Ebrahimi, Fahim; Koch, Marco; Dehghani, Faramarz

2012-10-01

Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone

PubMed Central

Wei, Qingshan; Qi, Hangfei; Luo, Wei; Tseng, Derek; Ki, So Jung; Wan, Zhe; Göröcs, Zoltán; Bentolila, Laurent A.; Wu, Ting-Ting; Sun, Ren; Ozcan, Aydogan

2014-01-01

Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments. PMID:24016065

Combining large area fluorescence with multiphoton microscopy for improved detection of oral epithelial neoplasia (Conference Presentation)

NASA Astrophysics Data System (ADS)

Pal, Rahul; Yang, Jinping; Qiu, Suimin; McCammon, Susan; Resto, Vicente; Vargas, Gracie

2016-03-01

Volumetric Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia representing the complex microstructural organization of mucosa, potentially providing high specificity for detection of neoplasia, but is limited by small imaging area. Large area fluorescence methods on the other hand show high sensitivity appropriate for screening but are hampered by low specificity. In this study, we apply MPAM-SHGM following guidance from large area fluorescence, by either autofluorescence or a targeted metabolic fluorophore, as a potentially clinically viable approach for detection of oral neoplasia. Sites of high neoplastic potentially were identified by large area red/green autofluorescence or by a fluorescently labelled deoxy-glucose analog, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to highlight areas of high glucose uptake across the buccal pouch of a hamster model for OSCC. Follow-up MPAM-SHGM was conducted on regions of interests (ROIs) to assess whether microscopy would reveal microscopic features associated with neoplasia to confirm or exclude large area fluorescence findings. Parameters for analysis included cytologic metrics, 3D epithelial connective tissue interface metrics (MPAM-SHGM) and intensity of fluorescence (widefield). Imaged sites were biopsied and processed for histology and graded by a pathologist. A small sample of human ex vivo tissues were also imaged. A generalized linear model combining image metrics from large area fluorescence and volumetric MPAM-SHGM indicated the ability to delineate normal and inflammation from neoplasia.

Active substrates improving sensitivity in biomedical fluorescence microscopy

NASA Astrophysics Data System (ADS)

Le Moal, E.; Leveque-Fort, S.; Fort, E.; Lacharme, J.-P.; Fontaine-Aupart, M.-P.; Ricolleau, C.

2005-08-01

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns.

PubMed

Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus

2015-08-25

We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

Simultaneous fluorescence and quantitative phase microscopy with single-pixel detectors

NASA Astrophysics Data System (ADS)

Liu, Yang; Suo, Jinli; Zhang, Yuanlong; Dai, Qionghai

2018-02-01

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

Resolution improvement by nonconfocal theta microscopy.

PubMed

Lindek, S; Stelzer, E H

1999-11-01

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.

In Vivo and Ex Vivo Confocal Microscopy for Dermatologic and Mohs Surgeons.

PubMed

Longo, Caterina; Ragazzi, Moira; Rajadhyaksha, Milind; Nehal, Kishwer; Bennassar, Antoni; Pellacani, Giovanni; Malvehy Guilera, Josep

2016-10-01

Confocal microscopy is a modern imaging device that has been extensively applied in skin oncology. More specifically, for tumor margin assessment, it has been used in two modalities: reflectance mode (in vivo on skin patient) and fluorescence mode (on freshly excised specimen). Although in vivo reflectance confocal microscopy is an add-on tool for lentigo maligna mapping, fluorescence confocal microscopy is far superior for basal cell carcinoma and squamous cell carcinoma margin assessment in the Mohs setting. This article provides a comprehensive overview of the use of confocal microscopy for skin cancer margin evaluation. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

PubMed Central

2013-01-01

Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules. PMID:23548159

Inducible fluorescent speckle microscopy

PubMed Central

Aguiar, Paulo; Belsley, Michael; Maiato, Helder

2016-01-01

The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

Inducible fluorescent speckle microscopy.

PubMed

Pereira, António J; Aguiar, Paulo; Belsley, Michael; Maiato, Helder

2016-01-18

The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. © 2016 Pereira et al.

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

Differential tissue expression of enhanced green fluorescent protein in 'green mice'.

PubMed

Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

2010-06-01

In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in 'green mice' from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these 'green mice' by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On immunohistochemical examination and on direct observation by confocal laser scanning microscopy, the level of EGFP expression varied among organs and tissues. EGFP expression was diffusely and strongly observed in the skin, pituitary, thyroid gland, parathyroid gland, heart, gall bladder, pancreas, adrenals and urinary bladder. There was only sporadic and weak expression of EGFP in the epithelium of the trachea, bronchus of the lung, stratified squamous epithelium and gastric glands of the stomach, hepatic bile ducts of the liver, glomeruli and renal tubules of the kidney and endo-metrial glands of the uterus. Furthermore, EGFP was only demonstrated within the goblet and paneth cells in the colon and small intestine, the tall columnar cells in the ductus epididymis, and the leydig cells in the testis. In conclusion, our results show that EGFP is differentially expressed in organs and tissues of 'green mice', which indicates that 'green mice' may prove useful for research involving transplantation and tissue clonality.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

PubMed Central

Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

2015-01-01

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses. PMID:25915529

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

PubMed

Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

2015-01-01

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

[Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

PubMed

Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

2010-02-01

This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another

PubMed Central

Kubilus, James K.; Zapater i Morales, Carolina; Linsenmayer, Thomas F.

2017-01-01

Purpose During development, the corneal epithelium (CE) and the conjunctiva are derived from the surface ectoderm. Here we have examined how, during development, the cells of these two issues become isolated from each other. Methods Epithelia from the anterior eyes of chicken embryos were labeled with the fluorescent, lipophilic dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). DiI was placed on the epithelial surface of the developing anterior eye and its diffusion was monitored by fluorescence microscopy. Concomitant morphologic changes in the surface cells of these epithelial were examined by scanning electron microscopy. Immunofluorescence was used to analyze the expression of cytokeratin K3, ZO-1, N-cadherin and Connexin-43 and the function of gap junctions was analyzed using a cut-loading with the fluorescent dye rhodamine-dextran. Results Prior to embryonic day 8 (E8), DiI placed on the surface of the CE spreads throughout all the epithelial cells of the anterior eye. When older eyes were similarly labeled, dye diffusion was restricted to the CE. Similarly, diffusion of DiI placed on the conjunctival surface after E8 was restricted to the conjunctiva. Scanning electron microscopy showed that developmentally (1) physical separations progressively form between the cells of the CE and those of the conjunctiva, and (2) by E8 these separations form a ring that completely encompasses the cornea. The functional restriction of gap junctions between these tissues did not occur until E14. Conclusions During ocular development, a barrier to the diffusion of DiI forms between the contiguous CE and conjunctiva prior to the differential expression of gap junctions within these tissues. PMID:28319640

Imaging bio-distribution of a topically applied dermatological cream on minipig skin using fluorescence lifetime imaging microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Alex, Aneesh; Chaney, Eric J.; Criley, Jennifer M.; Spillman, Darold R.; Hutchison, Phaedra B.; Li, Joanne; Marjanovic, Marina; Frey, Steve; Cook, Steven; Boppart, Stephen A.; Arp, Zane A.

2017-02-01

Currently there is a lack of in vivo techniques to evaluate the spatial bio-distribution of dermal drugs over time without the need to take multiple serial biopsies. To address this gap, we investigated the use of multi-photon optical imaging methods to non-invasively track drug distribution on miniature pig (Species: Sus scrofa, Strain: Göttingen) skin in vivo. Minipig skin is the standard comparative research model to human skin, and is anatomically and functionally similar. We employed fluorescence lifetime imaging microscopy (FLIM) to visualize the spatial distribution and residency time of a topically applied experimental dermatological cream. This was made possible by the endogenous fluorescent optical properties of the experimental drug (fluorescence lifetime > 3000 ps). Two different drug formulations were applied on 2 minipigs for 7 consecutive days, with the control creams applied on the contralateral side, followed by 7 days of post-application monitoring using a multi-modal optical imaging system (MPTflex-CARS, JenLab, Germany). FLIM images were obtained from the treated regions 24 hr post-application from day 1 to day 14 that allowed visualization of cellular and sub-cellular features associated with different dermal layers non-invasively to a depth of 200 µm. Five punch biopsies per animal were obtained from the corresponding treated regions between days 8 and 14 for bioanalytical analysis and comparison with results obtained using FLIM. In conclusion, utilization of non-invasive optical biopsy methods for dermal drug evaluation can provide true longitudinal monitoring of drug spatial distribution, remove sampling limitations, and be more time-efficient compared to traditional methods.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

PubMed Central

2013-01-01

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs. PMID:23388071

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.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Nucleocytoplasmic shuttling: the ins and outs of quantitative imaging.

PubMed

Molenaar, Chris; Weeks, Kate L

2018-05-17

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins.

PubMed

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

2016-10-01

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Fluorescent diamond nanoparticle as a probe of intracellular traffic in primary neurons in culture

NASA Astrophysics Data System (ADS)

Le, Xuan Loc; Lepagnol-Bestel, Aude-Marie; Adam, Marie-Pierre; Thomas, Alice; Dantelle, Géraldine; Chang, Cheng-Chun; Mohan, Nitin; Chang, Huan-Cheng; Treussart, François; Simonneau, Michel

2012-03-01

Neurons display dendritic spines plasticity and morphology anomalies in numerous psychiatric and neurodegenerative diseases. These changes are associated to abnormal dendritic traffic that can be evidenced by fluorescence microscopy. As a fluorescent probe we propose to use fluorescent diamond nanoparticles with size of < 50 nm. Color centers embedded inside the diamond nanoparticles are perfectly photostable emitters allowing for long-term tracking. Nanodiamond carbon surface is also well suited for biomolecule functionalization to target specific cellular compartments. We show that fluorescent nanodiamonds can be spontaneously internalized in neurons in culture and imaged by confocal and Total Internal Reflection (TIRF) microscopy with a high signal over background ratio.

Comparison of loop-mediated isothermal amplification assay and smear microscopy with culture for the diagnostic accuracy of tuberculosis.

PubMed

Gelaw, Baye; Shiferaw, Yitayal; Alemayehu, Marta; Bashaw, Abate Assefa

2017-01-17

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of death from infectious diseases worldwide. Sputum smear microscopy remains the most widely available pulmonary TB diagnostic tool particularly in resource limited settings. A highly sensitive diagnostic with minimal infrastructure, cost and training is required. Hence, we assessed the diagnostic performance of Loop-mediated isothermal amplification (LAMP) assay in detecting M.tuberculosis infection in sputum sample compared to LED fluorescent smear microscopy and culture. A cross-sectional study was conducted at the University of Gondar Hospital from June 01, 2015 to August 30, 2015. Pulmonary TB diagnosis using sputum LED fluorescence smear microscopy, TB-LAMP assay and culture were done. A descriptive analysis was used to determine demographic characteristics of the study participants. Analysis of sensitivity and specificity for smear microscopy and TB-LAMP compared with culture as a reference test was performed. Cohen's kappa was calculated as a measure of agreement between the tests. A total of 78 pulmonary presumptive TB patients sputum sample were analyzed. The overall sensitivity and specificity of LAMP were 75 and 98%, respectively. Among smear negative sputum samples, 33.3% sensitivity and 100% specificity of LAMP were observed. Smear microscopy showed 78.6% sensitivity and 98% specificity. LAMP and smear in series had sensitivity of 67.8% and specificity of 100%. LAMP and smear in parallel had sensitivity of 85.7% and specificity of 96%. The agreement between LAMP and fluorescent smear microscopy tests was very good (κ = 0.83, P-value ≤0.0001). TB-LAMP showed similar specificity but a slightly lower sensitivity with LED fluorescence microscopy. The specificity of LAMP and smear microscopy in series was high. The sensitivity of LAMP was insufficient for smear negative sputum samples.

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

2013-04-02

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

Functional photoacoustic microscopy of pH

NASA Astrophysics Data System (ADS)

Chatni, M. Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin I.; Wang, Lihong V.

2012-02-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

CARS microscopy of Alzheimer's diseased brain tissue

NASA Astrophysics Data System (ADS)

Enejder, Annika; Kiskis, Juris; Fink, Helen; Nyberg, Lena; Thyr, Jakob; Li, Jia-Yi

2014-02-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

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

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Simultaneous X-ray fluorescence and scanning X-ray diffraction microscopy at the Australian Synchrotron XFM beamline

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

Jones, Michael W. M.; Phillips, Nicholas W.; van Riessen, Grant A.

2016-08-11

Owing to its extreme sensitivity, quantitative mapping of elemental distributionsviaX-ray fluorescence microscopy (XFM) has become a key microanalytical technique. The recent realisation of scanning X-ray diffraction microscopy (SXDM) meanwhile provides an avenue for quantitative super-resolved ultra-structural visualization. The similarity of their experimental geometries indicates excellent prospects for simultaneous acquisition. Here, in both step- and fly-scanning modes, robust, simultaneous XFM-SXDM is demonstrated.

High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers

NASA Astrophysics Data System (ADS)

Siegel, Nisan; Lupashin, Vladimir; Storrie, Brian; Brooker, Gary

2016-12-01

Fresnel incoherent correlation holography (FINCH) microscopy is a promising approach for high-resolution biological imaging but has so far been limited to use with low-magnification, low-numerical-aperture configurations. We report the use of in-line incoherent interferometers made from uniaxial birefringent α-barium borate (α-BBO) or calcite crystals that overcome the aberrations and distortions present with previous implementations that employed spatial light modulators or gradient refractive index lenses. FINCH microscopy incorporating these birefringent elements and high-numerical-aperture oil immersion objectives could outperform standard wide-field fluorescence microscopy, with, for example, a 149 nm lateral point spread function at a wavelength of 590 nm. Enhanced resolution was confirmed with sub-resolution fluorescent beads. Taking the Golgi apparatus as a biological example, three different proteins labelled with GFP and two other fluorescent dyes in HeLa cells were resolved with an image quality that is comparable to similar samples captured by structured illumination microscopy.

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

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically-important Chrysomya rufifacies (Diptera: Calliphoridae)*

PubMed Central

Flores, Danielle; Miller, Amy L.; Showman, Angelique; Tobita, Caitlyn; Shimoda, Lori M.N.; Sung, Carl; Stokes, Alexander J.; Tomberlin, Jeffrey K.; Carter, David O.; Turner, Helen

2016-01-01

Entomological protocols for aging blow fly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analysing fly larvae differ, most rely on light microscopy, genetic analysis or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically-important blow fly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, non-bleaching, autofluorescence of larval posterior spiracles. High content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC. PMID:27706817

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

A new protocol for functional analysis of adipogenesis using reverse transfection technology and time-lapse video microscopy.

PubMed

Grönniger, Elke; Wessel, Sonja; Kühn, Sonja Christin; Söhle, Jörn; Wenck, Horst; Stäb, Franz; Winnefeld, Marc

2010-07-01

Since the worldwide increase in obesity represents a growing challenge for healthcare systems, research focusing on fat cell metabolism has become a focal point of interest. Here, we describe a small interfering RNA (siRNA)-technology-based screening method to study fat cell differentiation in human primary preadipocytes that could be further developed towards an automated middle-throughput screening procedure. First, we established optimal conditions for the reverse transfection of human primary preadipocytes demonstrating that an efficient reverse transfection of preadipocytes is technically feasible. Aligning the processes of reverse transfection and fat cell differentiation utilizing peroxisome proliferator-activated receptor gamma (PPAR gamma)-siRNA, we showed that preadipocyte differentiation was suppressed by knock-down of PPAR gamma, the key regulator of fat cell differentiation. The use of fluorescently labelled fatty acids in combination with fluorescence time-lapse microscopy over a longer period of time enabled us to quantify the PPAR gamma phenotype. Additionally, our data demonstrate that reverse transfection of human cultured preadipocytes with TIP60 (HIV-1 Tat-interacting protein 60)-siRNA lead to a TIP60 knock-down and subsequently inhibits fat cell differentiation, suggesting a role of this protein in human adipogenesis. In conclusion, we established a protocol that allows for an efficient functional and time-dependent analysis by quantitative time-lapse microscopy to identify novel adipogenesis-associated genes.

Detection, Mapping, and Quantification of Single Walled Carbon Nanotubes in Histological Specimens with Photoacoustic Microscopy

PubMed Central

Mikos, Antonios G.; Jansen, John A.; Shroyer, Kenneth R.; Wang, Lihong V.; Sitharaman, Balaji

2012-01-01

Aims In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Materials and Methods Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Results Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. Conclusions The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs. PMID:22496892

Fluorescence microscopy for measuring fibril angles in pine tracheids

Treesearch

Ralph O. Marts

1955-01-01

Observation and measurement of fibril angles in increment cores or similar small samples from living pine trees was facilitated by the use of fluorescence microscopy. Although some autofluorescence was present, brighter images could be obtained by staining the specimens with a 0.1% aqueous solution of a fluorochrome (Calcozine flavine TG extra concentrated, Calcozine...

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy

PubMed Central

Fernández, A.; Grüner-Nielsen, L.; Andreana, M.; Stadler, M.; Kirchberger, S.; Sturtzel, C.; Distel, M.; Zhu, L.; Kautek, W.; Leitgeb, R.; Baltuska, A.; Jespersen, K.; Verhoef, A.

2017-01-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy. PMID:28856032

Imaging intracellular protein dynamics by spinning disk confocal microscopy

PubMed Central

Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

2012-01-01

The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy.

PubMed

Fernández, A; Grüner-Nielsen, L; Andreana, M; Stadler, M; Kirchberger, S; Sturtzel, C; Distel, M; Zhu, L; Kautek, W; Leitgeb, R; Baltuska, A; Jespersen, K; Verhoef, A

2017-08-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.

Use of Complementary Approaches to Imaging Biomolecules and Endogenous and Exogenous Trace Elements and Nanoparticles in Biological Samples

NASA Astrophysics Data System (ADS)

Brown, Koshonna Dinettia

X-ray Fluorescence Microscopy (XFM) is a useful technique for study of biological samples. XFM was used to map and quantify endogenous biological elements as well as exogenous materials in biological samples, such as the distribution of titanium dioxide (TiO2) nanoparticles. TiO 2 nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic particles for cancer detection and treatment, drug delivery, and induction of DNA breaks. Delivery of such nanoparticles can be targeted to specific cells and subcellular structures. In this work, we develop two novel approaches to stain TiO2 nanoparticles for optical microscopy and to confirm that staining by XFM. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called CLICK chemistry, for labeling of azide conjugated TiO2 nanoparticles with "clickable" dyes such as alkyne Alexa Fluor dyes with a high fluorescent yield. To confirm that the optical fluorescence signals of nanoparticles stained in situ match the distribution of the Ti element, we used high resolution synchrotron X-Ray Fluorescence Microscopy (XFM) using the Bionanoprobe instrument at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific X-ray fluorescence showed excellent overlap with the location of Alexa Fluor optical fluorescence detected by confocal microscopy. In this work XFM was also used to investigate native elemental differences between two different types of head and neck cancer, one associated with human papilloma virus infection, the other virus free. Future work may see a cross between these themes, for example, exploration of TiO2 nanoparticles as anticancer treatment for these two different types of head and neck cancer.

Mueller matrix signature in advanced fluorescence microscopy imaging

NASA Astrophysics Data System (ADS)

Mazumder, Nirmal; Qiu, Jianjun; Kao, Fu-Jen; Diaspro, Alberto

2017-02-01

We have demonstrated the measurement and characterization of the polarization properties of a fluorescence signal using four-channel photon counting based Stokes-Mueller polarization microscopy. Thus, Lu-Chipman decomposition was applied to extract the critical polarization properties such as depolarization, linear retardance and the optical rotation of collagen type I fiber. We observed the spatial distribution of anisotropic and helical molecules of collagen from the reconstructed 2D Mueller images based on the fluorescence signal in a pixel-by-pixel manner.

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.

Determining absolute protein numbers by quantitative fluorescence microscopy.

PubMed

Verdaasdonk, Jolien Suzanne; Lawrimore, Josh; Bloom, Kerry

2014-01-01

Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition. © 2014 Elsevier Inc. All rights reserved.

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

PubMed Central

Wang, Xuhua; Yao, Sheng; Ahn, Hyo-Yang; Zhang, Yuanwei; Bondar, Mykhailo V.; Torres, Joseph A.; Belfield, Kevin D.

2010-01-01

Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated. PMID:21258480

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.

Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine.

PubMed

Alexiev, Ulrike; Volz, Pierre; Boreham, Alexander; Brodwolf, Robert

2017-07-01

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug-loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

NASA Astrophysics Data System (ADS)

Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

2009-10-01

Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification (LAMP) Assay for Tuberculosis Diagnosis in Adults with Chronic Cough in Malawi.

PubMed

Nliwasa, Marriott; MacPherson, Peter; Chisala, Palesa; Kamdolozi, Mercy; Khundi, McEwen; Kaswaswa, Kruger; Mwapasa, Mphatso; Msefula, Chisomo; Sohn, Hojoon; Flach, Clare; Corbett, Elizabeth L

2016-01-01

Current tuberculosis diagnostics lack sensitivity, and are expensive. Highly accurate, rapid and cheaper diagnostic tests are required for point of care use in low resource settings with high HIV prevalence. To investigate the sensitivity and specificity, and cost of loop-mediated isothermal amplification (LAMP) assay for tuberculosis diagnosis in adults with chronic cough compared to Xpert® MTB/RIF, fluorescence smear microscopy. Between October 2013 and March 2014, consecutive adults at a primary care clinic were screened for cough, offered HIV testing and assessed for tuberculosis using LAMP, Xpert® MTB/RIF and fluorescence smear microscopy. Sensitivity and specificity (with culture as reference standard), and costs were estimated. Of 273 adults recruited, 44.3% (121/273) were HIV-positive and 19.4% (53/273) had bacteriogically confirmed tuberculosis. The sensitivity of LAMP compared to culture was 65.0% (95% CI: 48.3% to 79.4%) with 100% (95% CI: 98.0% to 100%) specificity. The sensitivity of Xpert® MTB/RIF (77.5%, 95% CI: 61.5% to 89.2%) was similar to that of LAMP, p = 0.132. The sensitivity of concentrated fluorescence smear microscopy with routine double reading (87.5%, 95% CI: 73.2% to 95.8%) was higher than that of LAMP, p = 0.020. All three tests had high specificity. The lowest cost per test of LAMP was at batch size of 14 samples (US$ 9.98); this was lower than Xpert® MTB/RIF (US$ 13.38) but higher than fluorescence smear microscopy (US$ 0.65). The sensitivity of LAMP was similar to Xpert® MTB/RIF but lower than fluorescence smear microscopy; all three tests had high specificity. These findings support the Malawi policy that recommends a combination of fluorescence smear microscopy and Xpert® MTB/RIF prioritised for people living with HIV, already found to be smear-negative, or being considered for retreatment of tuberculosis.

Spectral characterization of Dictyostelium autofluorescence.

PubMed

Engel, Ruchira; Van Haastert, Peter J M; Visser, Antonie J W G

2006-03-01

Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from approximately 500 to 650 nm with a maximum at approximately 510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5-7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, pre-bleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium. Microsc. Res. Tech. 69:168-174, 2006. (c) 2006 Wiley-Liss, Inc.

Fluorescence microscope (Cyscope) for malaria diagnosis in pregnant women in Medani Hospital, Sudan.

PubMed

Hassan, Saad El-Din H; Haggaz, Abd Elrahium D; Mohammed-Elhassan, Ehab B; Malik, Elfatih M; Adam, Ishag

2011-09-24

Accuracy of diagnosis is the core for malaria control. Although microscopy is the gold standard in malaria diagnosis, its reliability is largely dependent on user skill. We compared performance of Cyscope fluorescence microscope with the Giemsa stained light microscopy for the diagnosis of malaria among pregnant women at Medani Hospital in Central Sudan. The area is characterized by unstable malaria transmission. Socio-demographic characteristics and obstetrics history were gathered using pre-tested questionnaires. Blood samples were collected from febrile pregnant women who were referred as malaria case following initial diagnosis by general microscopist. During the study period 128 febrile pregnant women presented at the hospital. Among them, Plasmodium falciparum malaria was detected in 82 (64.1%) and 80 (62.5%) by the Giemsa-stained light microscopy and the Cyscope fluorescence microscope, respectively. The sensitivity of the Cyscope fluorescence microscope was 97.6% (95% CI: 92.2%-99.6%). Out of 46 which were negative by Giemsa-stained light microscopy, 5 were positive by the Cyscope fluorescence microscope. This is translated in specificity of 89.1% (95% CI: 77.5%-95.9%). The positive and negative predictive value of Cyscope fluorescence microscope was 94.1% (95% CI: 87.4% -97.8%) and 95.3% (95% CI: 85.4% - 99.2%), respectively. This study has shown that Cyscope fluorescence microscope is a reliable diagnostic, sensitive and specific in diagnosing P. falciparum malaria among pregnant women in this setting. Further studies are needed to determine effectiveness in diagnosing other Plasmodium species and to compare it with other diagnostic tools e.g. rapid diagnostic tests and PCR.

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

USGS Publications Warehouse

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2017-03-27

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events

PubMed Central

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events. PMID:23823461

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

PubMed

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

In Situ Visualization of Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution Fluorescence Microscopy

PubMed Central

Boott, Charlotte E.; Laine, Romain F.; Mahou, Pierre; Finnegan, John R.; Leitao, Erin M.

2015-01-01

Abstract Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media. PMID:26477697

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

PubMed Central

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

2012-01-01

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

PubMed

Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

2012-11-01

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats

NASA Astrophysics Data System (ADS)

Thorling, Camilla A.; Liu, Xin; Burczynski, Frank J.; Fletcher, Linda M.; Gobe, Glenda C.; Roberts, Michael S.

2011-11-01

Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery. In orthotopic transplantation, the donor liver is exposed to periods of ischemia and when oxygenated blood is reintroduced to the liver, oxidative stress may develop and lead to graft failure. The aim of this project was to investigate whether noninvasive multiphoton and fluorescence lifetime imaging microscopy, without external markers, were useful in detecting early liver damage caused by I/R injury. Localized hepatic ischemia was induced in rats for 1 h followed by 4 h reperfusion. Multiphoton and fluorescence lifetime imaging microscopy was conducted prior to ischemia and up to 4 h of reperfusion and compared to morphological and biochemical assessment of liver damage. Liver function was significantly impaired at 2 and 4 h of reperfusion. Multiphoton microscopy detected liver damage at 1 h of reperfusion, manifested by vacuolated cells and heterogeneous spread of damage over the liver. The damage was mainly localized in the midzonal region of the liver acinus. In addition, fluorescence lifetime imaging showed a decrease in cellular metabolic activity. Multiphoton and fluorescence lifetime imaging microscopy detected evidence of early I/R injury both structurally and functionally. This provides a simple noninvasive technique useful for following progressive liver injury without external markers.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

PubMed

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-07-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

PubMed Central

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-01-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student. PMID:28686583

Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip.

PubMed

Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

2011-09-07

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes.

PubMed

Chitalia, Rhea; Mueller, Jenna; Fu, Henry L; Whitley, Melodi Javid; Kirsch, David G; Brown, J Quincy; Willett, Rebecca; Ramanujam, Nimmi

2016-09-01

Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.

Wide-Field Fluorescence Microscopy of Real-Time Bioconjugation Sensing

PubMed Central

Szalkowski, Marcin; Sulowska, Karolina; Grzelak, Justyna; Niedziółka-Jönsson, Joanna; Roźniecka, Ewa

2018-01-01

We apply wide-field fluorescence microscopy to measure real-time attachment of photosynthetic proteins to plasmonically active silver nanowires. The observation of this effect is enabled, on the one hand, by sensitive detection of fluorescence and, on the other hand, by plasmonic enhancement of protein fluorescence. We examined two sample configurations with substrates being a bare glass coverslip and a coverslip functionalized with a monolayer of streptavidin. The different preparation of the substrate changes the observed behavior as far as attachment of the protein is concerned as well as its subsequent photobleaching. For the latter substrate the conjugation process is measurably slower. The described method can be universally applied in studying protein-nanostructure interactions for real-time fluorescence-based sensing. PMID:29351211

Wide-Field Fluorescence Microscopy of Real-Time Bioconjugation Sensing.

PubMed

Szalkowski, Marcin; Sulowska, Karolina; Grzelak, Justyna; Niedziółka-Jönsson, Joanna; Roźniecka, Ewa; Kowalska, Dorota; Maćkowski, Sebastian

2018-01-19

We apply wide-field fluorescence microscopy to measure real-time attachment of photosynthetic proteins to plasmonically active silver nanowires. The observation of this effect is enabled, on the one hand, by sensitive detection of fluorescence and, on the other hand, by plasmonic enhancement of protein fluorescence. We examined two sample configurations with substrates being a bare glass coverslip and a coverslip functionalized with a monolayer of streptavidin. The different preparation of the substrate changes the observed behavior as far as attachment of the protein is concerned as well as its subsequent photobleaching. For the latter substrate the conjugation process is measurably slower. The described method can be universally applied in studying protein-nanostructure interactions for real-time fluorescence-based sensing.

Detecting Circular RNAs by RNA Fluorescence In Situ Hybridization.

PubMed

Zirkel, Anne; Papantonis, Argyris

2018-01-01

Fluorescence in situ hybridization (FISH) coupled to high-resolution microscopy is a powerful method for analyzing the subcellular localization of RNA. However, the detection of circular RNAs (circRNAs) using microscopy is challenging because the only feature of a circRNA that can be used for the probe design is its junction. Circular RNAs are expressed at varying levels, and for their efficient monitoring by FISH, background fluorescence levels need to be kept low. Here, we describe a FISH protocol coupled to high-precision localizations using a single fluorescently labeled probe spanning the circRNA junction; this allows circRNA detection in mammalian cells with high signal-to-noise ratios.

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.

Multiphoton fluorescence imaging of NADH to quantify metabolic changes in epileptic tissue in vitro

NASA Astrophysics Data System (ADS)

Chia, Thomas H.; Zinter, Joseph; Spencer, Dennis D.; Williamson, Anne; Levene, Michael J.

2007-02-01

A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD +; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

PubMed

Braaf, Boy; de Boer, Johannes F

2017-03-20

Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

Widely accessible method for superresolution fluorescence imaging of living systems

PubMed Central

Dedecker, Peter; Mo, Gary C. H.; Dertinger, Thomas; Zhang, Jin

2012-01-01

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule–based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging. PMID:22711840

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

PubMed

Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2015-03-01

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structured illumination microscopy for dual-modality 3D sub-diffraction resolution fluorescence and refractive-index reconstruction

PubMed Central

Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

2017-01-01

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction resolution coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate together the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction resolution fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin. This demonstration may suggest SI as an especially promising imaging technique to enable future biological studies that explore synergistically operating biophysical/biochemical and molecular mechanisms at sub-diffraction resolutions. PMID:29296504

An ensemble and single-molecule fluorescence microscopy investigation of phase-separated monolayer films stained with Nile Red.

PubMed

Lu, Yin; Porterfield, Robyn; Thunder, Terri; Paige, Matthew F

2011-01-01

Phase-separated Langmuir-Blodgett monolayer films prepared from mixtures of arachidic acid (C19H39COOH) and perfluorotetradecanoic acid (C13F27COOH) were stained via spin-casting with the polarity sensitive phenoxazine dye Nile Red, and characterized using a combination of ensemble and single-molecule fluorescence microscopy measurements. Ensemble fluorescence microscopy and spectromicroscopy showed that Nile Red preferentially associated with the hydrogenated domains of the phase-separated films, and was strongly fluorescent in these areas of the film. These measurements, in conjunction with single-molecule fluorescence imaging experiments, also indicated that a small sub-population of dye molecules localizes on the perfluorinated regions of the sample, but that this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. The relative importance of selective dye adsorption and local polarity sensitivity of Nile Red for staining applications in phase-separated LB films as well as in cellular environments is discussed in context of the experimental results. Copyright © 2010 Elsevier B.V. All rights reserved.

Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

PubMed

Rizk, Aurélien; Paul, Grégory; Incardona, Pietro; Bugarski, Milica; Mansouri, Maysam; Niemann, Axel; Ziegler, Urs; Berger, Philipp; Sbalzarini, Ivo F

2014-03-01

Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires <1 min and <5 min for 2D and 3D images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

PubMed

Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

2012-08-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Automatic tracking of cells for video microscopy in patch clamp experiments.

PubMed

Peixoto, Helton M; Munguba, Hermany; Cruz, Rossana M S; Guerreiro, Ana M G; Leao, Richardson N

2014-06-20

Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.

PubMed

Klohs, Jan; Gräfe, Michael; Graf, Kristof; Steinbrink, Jens; Dietrich, Thore; Stibenz, Dietger; Bahmani, Peyman; Kronenberg, Golo; Harms, Christoph; Endres, Matthias; Lindauer, Ute; Greger, Klaus; Stelzer, Ernst H K; Dirnagl, Ulrich; Wunder, Andreas

2008-10-01

Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Intravital multiphoton fluorescence imaging and optical manipulation of spinal cord in mice, using a compact fiber laser system.

PubMed

Oshima, Yusuke; Horiuch, Hideki; Honkura, Naoki; Hikita, Atsuhiko; Ogata, Tadanori; Miura, Hiromasa; Imamura, Takeshi

2014-09-01

Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future. © 2014 Wiley Periodicals, Inc.

Rapid and easy identification of Illicium verum Hook. f. and its adulterant Illicium anisatum Linn. by fluorescent microscopy and gas chromatography.

PubMed

Joshi, Vaishali C; Srinivas, Pullela V; Khan, Ikhlas A

2005-01-01

Illicium verum Hook. f. is used as an herbal tea to treat colic pain in infants. Reports suggest that Star anise herbal tea may be adulterated with Illicium anisatum Linn. A short and rapid method using microscopy and gas chromatography (GC) was developed to detect I. anisatum Linn., an adulterant in the powdered mixture of I. verum. Anatomical differences in the epicarp cells of I. verum and I. anisatum fruits were clearly defined as examined under fluorescent microscopy and scanning electron microscopy. A GC method was developed for quick identification of possible I. anisatum adulteration with I. verum.

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

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

[Assessment of a rapid diagnostic test and portable fluorescent microscopy for malaria diagnosis in Cotonou (Bénin)].

PubMed

Ogouyèmi-Hounto, A; Kinde-Gazard, D; Keke, C; Gonçalves, E; Alapini, N; Adjovi, F; Adisso, L; Bossou, C; Denon, Y V; Massougbodji, A

2013-02-01

The aim of the study was to determine the accuracy of a rapid diagnostic test (SD Bioline Malaria Ag P.f/ Pan®) and fluorescent microscopy (CyScope®) in confirming presumptive malaria diagnosis in Cotonou. Thick blood smear was used as the reference technique for comparison. Testing was conducted on persons between the ages of 6 months and 70 years at two hospitals from June to October 2010. If malaria was suspected in the sample by the nurse based on clinical findings and sent to laboratory for confirmation, one thick smear, one rapid diagnostic test and one slide for the fluorescent microscopy were performed. All tests were read in hospital laboratories involved with the quality control of thick blood smear in the parasitology laboratory of National University Hospital of Cotonou. A total of 354 patients with clinical diagnosis of malaria were included. Malaria prevalence determined by thick smear, rapid diagnostic test and fluorescent microscopy was 22.8%, 25.4%, and 25.1% respectively. The sensitivity, specificity, positive and negative predictive values compared to the thick smears were 96.3, 95.6, 86.7, and 98.9% for rapid diagnostic test; and 97.5, 96.7, 89.8, and 99.27% for fluorescent microscopy. With these performances, these tests meet acceptability standards recommended by WHO for rapid tests (sensitivity > 95%). These two methods have advantages for the confirmation of malaria diagnosis in peripheral health structures that lack the resources to conduct diagnosis confirmation by the thick blood smear.

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

PubMed

Jia, Peng-Fei; Li, Hong-Ju; Yang, Wei-Cai

2017-01-01

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

PubMed Central

Afshar, Yaser; Sbalzarini, Ivo F.

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

Single-molecule microscopy reveals membrane microdomain organization of cells in a living vertebrate.

PubMed

Schaaf, Marcel J M; Koopmans, Wiepke J A; Meckel, Tobias; van Noort, John; Snaar-Jagalska, B Ewa; Schmidt, Thomas S; Spaink, Herman P

2009-08-19

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.

Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

PubMed Central

Boehm, Elizabeth M.; Subramanyam, Shyamal; Ghoneim, Mohamed; Washington, M. Todd; Spies, Maria

2016-01-01

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary, quaternary, etc.) complexes using multi-color setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches has become today. PMID:27793278

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

PubMed

Afshar, Yaser; Sbalzarini, Ivo F

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations

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

Yamada, Shigeyuki; Zhang Xiuquan; Kadono, Toshie

Aims: Our goal was to determine if clinically relevant concentrations of aqueous extract of cigarette smoke (CSE) have direct deleterious effects on ventricular myocytes during simulated ischemia, and to investigate the mechanisms involved. Methods: CSE was prepared with a smoking chamber. Ischemia was simulated by metabolic inhibition (MI) with cyanide (CN) and 0 glucose. Adult rabbit and mouse ventricular myocyte [Ca{sup 2+}]{sub i} was measured by flow cytometry using fluo-3. Mitochondrial [Ca{sup 2+}] was measured with confocal microscopy, and Rhod-2 fluorescence. The mitochondrial permeability transition (MPT) was detected by TMRM fluorescence and myocyte contracture. Myocyte oxidative stress was quantified bymore » dichlorofluorescein (DCF) fluorescence with confocal microscopy. Results: CSE 0.1% increased myocyte contracture caused by MI. The nicotine concentration (HPLC) in 0.1% CSE was 15 ng/ml, similar to that in humans after smoking cigarettes. CSE 0.1% increased mitochondrial Ca{sup 2+} uptake, and increased the susceptibility of mitochondria to the MPT. CSE 0.1% increased DCF fluorescence in isolated myocytes, and increased [Ca{sup 2+}]{sub i} in paced myocytes exposed to 2.0 mM CN, 0 glucose (P-MI). These effects were inhibited by the superoxide scavenger Tiron. The effect of CSE on [Ca{sup 2+}]{sub i} during P-MI was also prevented by ranolazine. Conclusions: CSE in clinically relevant concentrations increases myocyte [Ca{sup 2+}]{sub i} during simulated ischemia, and increases myocyte susceptibility to the MPT. These effects appear to be mediated at least in part by oxidative radicals in CSE, and likely contribute to the effects of cigarette smoke to increase myocardial infarct size, and to decrease angina threshold.« less

Electrochemical Glucose Biosensor of Platinum Nanospheres Connected by Carbon Nanotubes

PubMed Central

Claussen, Jonathan C.; Kim, Sungwon S.; Haque, Aeraj ul; Artiles, Mayra S.; Porterfield, D. Marshall; Fisher, Timothy S.

2010-01-01

Background Glucose biosensors comprised of nanomaterials such as carbon nanotubes (CNTs) and metallic nanoparticles offer enhanced electrochemical performance that produces highly sensitive glucose sensing. This article presents a facile biosensor fabrication and biofunctionalization procedure that utilizes CNTs electrochemically decorated with platinum (Pt) nanospheres to sense glucose amperometrically with high sensitivity. Method Carbon nanotubes are grown in situ by microwave plasma chemical vapor deposition (MPCVD) and electro-chemically decorated with Pt nanospheres to form a CNT/Pt nanosphere composite biosensor. Carbon nanotube electrodes are immobilized with fluorescently labeled bovine serum albumin (BSA) and analyzed with fluorescence microscopy to demonstrate their biocompatibility. The enzyme glucose oxidase (GOX) is immobilized onto the CNT/Pt nanosphere biosensor by a simple drop-coat method for amperometric glucose sensing. Results Fluorescence microscopy demonstrates the biofunctionalization capability of the sensor by portraying adsorption of fluorescently labeled BSA unto MPCVD-grown CNT electrodes. The subsequent GOX–CNT/Pt nanosphere biosensor demonstrates a high sensitivity toward H2O2 (7.4 μA/mM/cm2) and glucose (70 μA/mM/cm2), with a glucose detection limit and response time of 380 nM (signal-to-noise ratio = 3) and 8 s (t90%), respectively. The apparent Michaelis–Menten constant (0.64 mM) of the biosensor also reflects the improved sensitivity of the immobilized GOX/nanomaterial complexes. Conclusions The GOX–CNT/Pt nanosphere biosensor outperforms similar CNT, metallic nanoparticle, and more conventional carbon-based biosensors in terms of glucose sensitivity and detection limit. The biosensor fabrication and biofunctionalization scheme can easily be scaled and adapted for microsensors for physiological research applications that require highly sensitive glucose sensing. PMID:20307391

Chemistry Is Dead. Long Live Chemistry!

PubMed

Lavis, Luke D

2017-10-03

Chemistry, once king of fluorescence microscopy, was usurped by the field of fluorescent proteins. The increased demands of modern microscopy techniques on the "photon budget" require better and brighter fluorophores, causing a renewed interest in synthetic dyes. Here, we review the recent advances in biochemistry, protein engineering, and organic synthesis that have allowed a triumphant return of chemical fluorophores to modern biological imaging.

Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein; Geral I. McDonald; Kathiravetpillai Arumuganathan

2001-01-01

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids...

Synchrotron-based X-ray Fluorescence Microscopy in Conjunction with Nanoindentation to Study Molecular-Scale Interactions of Phenol–Formaldehyde in Wood Cell Walls

Treesearch

Joseph E. Jakes; Christopher G. Hunt; Daniel J. Yelle; Linda Lorenz; Kolby Hirth; Sophie-Charlotte Gleber; Stefan Vogt; Warren Grigsby; Charles R. Frihart

2015-01-01

Understanding and controlling molecular-scale interactions between adhesives and wood polymers are critical to accelerate the development of improved adhesives for advanced wood-based materials. The submicrometer resolution of synchrotron-based X-ray fluorescence microscopy (XFM) was found capable of mapping and quantifying infiltration of Br-labeled phenol−...

Analysis of multi-channel microscopy: Spectral self-interference, multi-detector confocal and 4Pi systems

NASA Astrophysics Data System (ADS)

Davis, Brynmor J.

Fluorescence microscopy is an important and ubiquitous tool in biological imaging due to the high specificity with which fluorescent molecules can be attached to an organism and the subsequent nondestructive in-vivo imaging allowed. Focused-light microscopies allow three-dimensional fluorescence imaging but their resolution is restricted by diffraction. This effect is particularly limiting in the axial dimension as the diffraction-limited focal volume produced by a lens is more extensive along the optical axis than perpendicular to it. Approaches such as confocal microscopy and 4Pi microscopy have been developed to improve the axial resolution. Spectral Self-Interference Fluorescence Microscopy (SSFM) is another high-axial-resolution technique and is the principal subject of this dissertation. Nanometer-precision localization of a single fluorescent layer has been demonstrated using SSFM. This accuracy compares favorably with the axial resolutions given by confocal and 4Pi systems at similar operating parameters (these resolutions are approximately 350nm and 80nm respectively). This theoretical work analyzes the expected performance of the SSFM system when imaging a general object, i.e. an arbitrary fluorophore density function rather than a single layer. An existing model of SSFM is used in simulations to characterize the system's resolution. Several statistically-based reconstruction methods are applied to show that the expected resolution for SSFM is similar to 4Pi microscopy for a general object but does give very high localization accuracy when the object is known to consist of a limited number of layers. SSFM is then analyzed in a linear systems framework and shown to have strong connections, both physically and mathematically, to a multi-channel 4Pi microscope. Fourier-domain analysis confirms that SSFM cannot be expected to outperform this multi-channel 4Pi instrument. Differences between the channels in spatial-scanning, multi-channel microscopies are then exploited to show that such instruments can operate at a sub-Nyquist scanning rate but still produce images largely free of aliasing effects. Multi-channel analysis is also used to show how light typically discarded in confocal and 4Pi systems can be collected and usefully incorporated into the measured image.

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.

FRET-FLIM microscopy

NASA Astrophysics Data System (ADS)

Elangovan, Masilamani; Day, Richard N.; Periasamy, Ammasi

2002-06-01

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy coupled with the development of new fluorescent probes provide the tools to study protein interactions in living specimens. Spectral bleed-through or cross talk is a problem in one- and two-photon microscopy to recognize whether one is observing the sensitized emission or the bleed-through signals. In contrast, FLIM (fluorescence lifetime imaging microscopy) or lifetime measurements are independent of excitation intensity or fluorophore concentration. The combination of FLIM and FRET will provide high spatial (nanometer) and temporal (nanoseconds) resolution when compared to steady state FRET imaging. Importantly, spectral bleed-through is not an issue in FLIM imaging because only the donor fluorophore lifetime is measured. The presence of acceptor molecules within the local environment of the donor that permit energy transfer will influence the fluorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the FRET efficiency and the distance between donor- and acceptor-labeled proteins. Moreover, the FRET-FLIM technique allows monitoring more than one pair of protein interactions in a single living cell.

Thin-film tunable filters for hyperspectral fluorescence microscopy

PubMed Central

Favreau, Peter; Hernandez, Clarissa; Lindsey, Ashley Stringfellow; Alvarez, Diego F.; Rich, Thomas; Prabhat, Prashant

2013-01-01

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes. PMID:24077519

Dendrimer-assisted patch-clamp sizing of nuclear pores

PubMed Central

Bustamante, J.O.; Michelette, E.R.F.; Geibel, J.P.; Hanover, J.A.; McDonnell, T.J.; Dean, D.A.

2015-01-01

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be ≅10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5–10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Star-burst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions. PMID:10784359

Comparison of flow cytometry, fluorescence microscopy and spectrofluorometry for analysis of gene electrotransfer efficiency.

PubMed

Marjanovič, Igor; Kandušer, Maša; Miklavčič, Damijan; Keber, Mateja Manček; Pavlin, Mojca

2014-12-01

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.

Aberrations and adaptive optics in super-resolution microscopy.

PubMed

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-08-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy - or rather nanoscopy - to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

PubMed

Wille, Guillaume; Hellal, Jennifer; Ollivier, Patrick; Richard, Annie; Burel, Agnes; Jolly, Louis; Crampon, Marc; Michel, Caroline

2017-12-01

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Sub-micron scale patterning of fluorescent silver nanoclusters using low-power laser.

PubMed

Kunwar, Puskal; Hassinen, Jukka; Bautista, Godofredo; Ras, Robin H A; Toivonen, Juha

2016-04-05

Noble metal nanoclusters are ultrasmall nanomaterials with tunable properties and huge application potential; however, retaining their enhanced functionality is difficult as they readily lose their properties without stabilization. Here, we demonstrate a facile synthesis of highly photostable silver nanoclusters in a polymer thin film using visible light photoreduction. Furthermore, the different stages of the nanocluster formation are investigated in detail using absorption and fluorescence spectroscopy, fluorescence microscopy, and atomic force microscopy. A cost-effective fabrication of photostable micron-sized fluorescent silver nanocluster barcode is demonstrated in silver-impregnated polymer films using a low-power continuous-wave laser diode. It is shown that a laser power of as low as 0.75 mW is enough to write fluorescent structures, corresponding to the specifications of a commercially available laser pointer. The as-formed nanocluster-containing microstructures can be useful in direct labeling applications such as authenticity marking and fluorescent labeling.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

Photobleaching correction in fluorescence microscopy images

NASA Astrophysics Data System (ADS)

Vicente, Nathalie B.; Diaz Zamboni, Javier E.; Adur, Javier F.; Paravani, Enrique V.; Casco, Víctor H.

2007-11-01

Fluorophores are used to detect molecular expression by highly specific antigen-antibody reactions in fluorescence microscopy techniques. A portion of the fluorophore emits fluorescence when irradiated with electromagnetic waves of particular wavelengths, enabling its detection. Photobleaching irreversibly destroys fluorophores stimulated by radiation within the excitation spectrum, thus eliminating potentially useful information. Since this process may not be completely prevented, techniques have been developed to slow it down or to correct resulting alterations (mainly, the decrease in fluorescent signal). In the present work, the correction by photobleaching curve was studied using E-cadherin (a cell-cell adhesion molecule) expression in Bufo arenarum embryos. Significant improvements were observed when applying this simple, inexpensive and fast technique.

Tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism.

PubMed

Cruz, Luis J; Tacken, Paul J; Zeelenberg, Ingrid S; Srinivas, Mangala; Bonetto, Fernando; Weigelin, Bettina; Eich, Christina; de Vries, I Jolanda; Figdor, Carl G

2014-12-01

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), involved in the induction of immunity and currently exploited for antitumor immunotherapies. An optimized noninvasive imaging modality capable of determining and quantifying DC-targeted nanoparticle (NP) trajectories could provide valuable information regarding therapeutic vaccine outcome. Here, targeted poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) recognizing DC receptors were equipped with superparamagnetic iron oxide particles (SPIO) or gold nanoparticles with fluorescently labeled antigen. The fluorescent label allowed for rapid analysis and quantification of DC-specific uptake of targeted PLGA NPs in comparison to uptake by other cells. Transmission electron microscopy (TEM) showed that a fraction of the encapsulated antigen reached the lysosomal compartment of DCs, where SPIO and gold were already partially released. However, part of the PLGA NPs localized within the cytoplasm, as confirmed by confocal microscopy. DCs targeted with NPs carrying SPIO or fluorescent antigen were detected within lymph nodes as early as 1 h after injection by magnetic resonance imaging (MRI). Despite the fact that targeting did not markedly affect PLGA NP biodistribution on organism and tissue level, it increased delivery of NPs to DCs residing in peripheral lymph nodes and resulted in enhanced T cell proliferation. In conclusion, two imaging agents within a single carrier allows tracking of targeted PLGA NPs at the subcellular, cellular, and organismal levels, thereby facilitating the rational design of in vivo targeted vaccination strategies.

Inflammation Modulates Murine Venous Thrombosis Resolution In Vivo: Assessment by Multimodal Fluorescence Molecular Imaging

PubMed Central

Ripplinger, Crystal M.; Kessinger, Chase W.; Li, Chunqiang; Kim, Jin Won; McCarthy, Jason R.; Weissleder, Ralph; Henke, Peter K.; Lin, Charles P.; Jaffer, Farouc A.

2012-01-01

Objective Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here we develop and evaluate two integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture, and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo. Methods and Results Murine DVT were created with topical 5% FeCl3 application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase (MMP) activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy (IVM). Day 4 analyses showed robust relationships among in vivo thrombus macrophages, MMP activity, and FITC-dextran deposition (r>0.70, p<0.01). In a serial two-timepoint study, mice with DVT underwent IVM at day 4 and at day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (p<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography (FMT-CT) was employed, and detected macrophages within jugular DVT (p<0.05 vs. sham-controls). Conclusions Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo, and can inform the magnitude of thrombus resolution. PMID:22995524

Non-invasive near-infrared fluorescence imaging of the neutrophil response in a mouse model of transient cerebral ischaemia.

PubMed

Vaas, Markus; Enzmann, Gaby; Perinat, Therese; Siler, Ulrich; Reichenbach, Janine; Licha, Kai; Kipar, Anja; Rudin, Markus; Engelhardt, Britta; Klohs, Jan

2017-08-01

Near-infrared fluorescence (NIRF) imaging enables non-invasive monitoring of molecular and cellular processes in live animals. Here we demonstrate the suitability of NIRF imaging to investigate the neutrophil response in the brain after transient middle cerebral artery occlusion (tMCAO). We established procedures for ex vivo fluorescent labelling of neutrophils without affecting their activation status. Adoptive transfer of labelled neutrophils in C57BL/6 mice before surgery resulted in higher fluorescence intensities over the ischaemic hemisphere in tMCAO mice with NIRF imaging when compared with controls, corroborated by ex vivo detection of labelled neutrophils using fluorescence microscopy. NIRF imaging showed that neutrophils started to accumulate immediately after tMCAO, peaking at 18 h, and were still visible until 48 h after reperfusion. Our data revealed accumulation of neutrophils also in extracranial tissue, indicating damage in the external carotid artery territory in the tMCAO model. Antibody-mediated inhibition of α4-integrins did reduce fluorescence signals at 18 and 24, but not at 48 h after reperfusion, compared with control treatment animals. Antibody treatment reduced cerebral lesion volumes by 19%. In conclusion, the non-invasive nature of NIRF imaging allows studying the dynamics of neutrophil recruitment and its modulation by targeted interventions in the mouse brain after transient experimental cerebral ischaemia.

Non-invasive near-infrared fluorescence imaging of the neutrophil response in a mouse model of transient cerebral ischaemia

PubMed Central

Vaas, Markus; Enzmann, Gaby; Perinat, Therese; Siler, Ulrich; Reichenbach, Janine; Licha, Kai; Kipar, Anja; Rudin, Markus; Engelhardt, Britta

2016-01-01

Near-infrared fluorescence (NIRF) imaging enables non-invasive monitoring of molecular and cellular processes in live animals. Here we demonstrate the suitability of NIRF imaging to investigate the neutrophil response in the brain after transient middle cerebral artery occlusion (tMCAO). We established procedures for ex vivo fluorescent labelling of neutrophils without affecting their activation status. Adoptive transfer of labelled neutrophils in C57BL/6 mice before surgery resulted in higher fluorescence intensities over the ischaemic hemisphere in tMCAO mice with NIRF imaging when compared with controls, corroborated by ex vivo detection of labelled neutrophils using fluorescence microscopy. NIRF imaging showed that neutrophils started to accumulate immediately after tMCAO, peaking at 18 h, and were still visible until 48 h after reperfusion. Our data revealed accumulation of neutrophils also in extracranial tissue, indicating damage in the external carotid artery territory in the tMCAO model. Antibody-mediated inhibition of α4-integrins did reduce fluorescence signals at 18 and 24, but not at 48 h after reperfusion, compared with control treatment animals. Antibody treatment reduced cerebral lesion volumes by 19%. In conclusion, the non-invasive nature of NIRF imaging allows studying the dynamics of neutrophil recruitment and its modulation by targeted interventions in the mouse brain after transient experimental cerebral ischaemia. PMID:27789786

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Multimodal nonlinear imaging of arabidopsis thaliana root cell

NASA Astrophysics Data System (ADS)

Jang, Bumjoon; Lee, Sung-Ho; Woo, Sooah; Park, Jong-Hyun; Lee, Myeong Min; Park, Seung-Han

2017-07-01

Nonlinear optical microscopy has enabled the possibility to explore inside the living organisms. It utilizes ultrashort laser pulse with long wavelength (greater than 800nm). Ultrashort pulse produces high peak power to induce nonlinear optical phenomenon such as two-photon excitation fluorescence (TPEF) and harmonic generations in the medium while maintaining relatively low average energy pre area. In plant developmental biology, confocal microscopy is widely used in plant cell imaging after the development of biological fluorescence labels in mid-1990s. However, fluorescence labeling itself affects the sample and the sample deviates from intact condition especially when labelling the entire cell. In this work, we report the dynamic images of Arabidopsis thaliana root cells. This demonstrates the multimodal nonlinear optical microscopy is an effective tool for long-term plant cell imaging.

How neurosecretory vesicles release their cargo.

PubMed

Scalettar, Bethe A

2006-04-01

Neurons and related cell types often contain two major classes of neurosecretory vesicles, synaptic vesicles (SVs) and dense-core granules (DCGs), which store and release distinct cargo. SVs store and release classic neurotransmitters, which facilitate propagation of action potentials across the synaptic cleft, whereas DCGs transport, store, and release hormones, proteins, and neuropeptides, which facilitate neuronal survival, synaptic transmission, and learning. Over the past few years, there has been a major surge in our understanding of many of the key molecular mechanisms underlying cargo release from SVs and DCGs. This surge has been driven largely by the use of fluorescence microscopy (especially total internal reflection fluorescence microscopy) to visualize SVs or DCGs in living cells. This review highlights some of the recent insights into cargo release from neurosecretory vesicles provided by fluorescence microscopy, with emphasis on DCGs.

Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

PubMed

Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

2013-07-01

Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

Adaptive Spot Detection With Optimal Scale Selection in Fluorescence Microscopy Images.

PubMed

Basset, Antoine; Boulanger, Jérôme; Salamero, Jean; Bouthemy, Patrick; Kervrann, Charles

2015-11-01

Accurately detecting subcellular particles in fluorescence microscopy is of primary interest for further quantitative analysis such as counting, tracking, or classification. Our primary goal is to segment vesicles likely to share nearly the same size in fluorescence microscopy images. Our method termed adaptive thresholding of Laplacian of Gaussian (LoG) images with autoselected scale (ATLAS) automatically selects the optimal scale corresponding to the most frequent spot size in the image. Four criteria are proposed and compared to determine the optimal scale in a scale-space framework. Then, the segmentation stage amounts to thresholding the LoG of the intensity image. In contrast to other methods, the threshold is locally adapted given a probability of false alarm (PFA) specified by the user for the whole set of images to be processed. The local threshold is automatically derived from the PFA value and local image statistics estimated in a window whose size is not a critical parameter. We also propose a new data set for benchmarking, consisting of six collections of one hundred images each, which exploits backgrounds extracted from real microscopy images. We have carried out an extensive comparative evaluation on several data sets with ground-truth, which demonstrates that ATLAS outperforms existing methods. ATLAS does not need any fine parameter tuning and requires very low computation time. Convincing results are also reported on real total internal reflection fluorescence microscopy images.

Rare-earth doped nanocomposites enable multiscale targeted short-wave infrared imaging of metastatic breast cancer

NASA Astrophysics Data System (ADS)

Pierce, Mark C.; Higgins, Laura M.; Ganapathy, Vidya; Kantamneni, Harini; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.

2017-02-01

We are investigating the ability of targeted rare earth (RE) doped nanocomposites to detect and track micrometastatic breast cancer lesions to distant sites in pre-clinical in vivo models. Functionalizing RE nanocomposites with AMD3100 promotes targeting to CXCR4, a recognized marker for highly metastatic disease. Mice were inoculated with SCP-28 (CXCR4 positive) and 4175 (CXCR4 negative) cell lines. Whole animal in vivo SWIR fluorescence imaging was performed after bioluminescence imaging confirmed tumor burden in the lungs. Line-scanning confocal fluorescence microscopy provided high-resolution imaging of RE nanocomposite uptake and native tissue autofluorescence in ex vivo lung specimens. Co-registered optical coherence tomography imaging allowed assessment of tissue microarchitecture. In conclusion, multiscale optical molecular imaging can be performed in pre-clinical models of metastatic breast cancer, using targeted RE-doped nanocomposites.

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Tracking of cell nuclei for assessment of in vitro uptake kinetics in ultrasound-mediated drug delivery using fibered confocal fluorescence microscopy.

PubMed

Derieppe, Marc; de Senneville, Baudouin Denis; Kuijf, Hugo; Moonen, Chrit; Bos, Clemens

2014-10-01

Previously, we demonstrated the feasibility to monitor ultrasound-mediated uptake of a cell-impermeable model drug in real time with fibered confocal fluorescence microscopy. Here, we present a complete post-processing methodology, which corrects for cell displacements, to improve the accuracy of pharmacokinetic parameter estimation. Nucleus detection was performed based on the radial symmetry transform algorithm. Cell tracking used an iterative closest point approach. Pharmacokinetic parameters were calculated by fitting a two-compartment model to the time-intensity curves of individual cells. Cells were tracked successfully, improving time-intensity curve accuracy and pharmacokinetic parameter estimation. With tracking, 93 % of the 370 nuclei showed a fluorescence signal variation that was well-described by a two-compartment model. In addition, parameter distributions were narrower, thus increasing precision. Dedicated image analysis was implemented and enabled studying ultrasound-mediated model drug uptake kinetics in hundreds of cells per experiment, using fiber-based confocal fluorescence microscopy.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

PubMed

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

PubMed

Müller, Andreas; Neukam, Martin; Ivanova, Anna; Sönmez, Anke; Münster, Carla; Kretschmar, Susanne; Kalaidzidis, Yannis; Kurth, Thomas; Verbavatz, Jean-Marc; Solimena, Michele

2017-02-02

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage

NASA Astrophysics Data System (ADS)

Wachsmann-Hogiu, Sebastian; Krakow, Deborah; Kirilova, Veneta T.; Cohn, Daniel H.; Bertolotto, Cristina; Acuna, Dora; Fang, Qiyin; Krivorov, Nikola; Farkas, Daniel L.

2005-03-01

It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

PubMed

Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

2011-03-01

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

Analysis of gene expression levels in individual bacterial cells without image segmentation.

PubMed

Kwak, In Hae; Son, Minjun; Hagen, Stephen J

2012-05-11

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly. Copyright © 2012 Elsevier Inc. All rights reserved.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

PubMed

Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

2016-03-18

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

In vivo analysis of intestinal permeability following hemorrhagic shock

PubMed Central

Alsaigh, Tom; Chang, Marisol; Richter, Michael; Mazor, Rafi; Kistler, Erik B

2015-01-01

AIM: To determine the time course of intestinal permeability changes to proteolytically-derived bowel peptides in experimental hemorrhagic shock. METHODS: We injected fluorescently-conjugated casein protein into the small bowel of anesthetized Wistar rats prior to induction of experimental hemorrhagic shock. These molecules, which fluoresce when proteolytically cleaved, were used as markers for the ability of proteolytically cleaved intestinal products to access the central circulation. Blood was serially sampled to quantify the relative change in concentration of proteolytically-cleaved particles in the systemic circulation. To provide spatial resolution of their location, particles in the mesenteric microvasculature were imaged using in vivo intravital fluorescent microscopy. The experiments were then repeated using an alternate measurement technique, fluorescein isothiocyanate (FITC)-labeled dextrans 20, to semi-quantitatively verify the ability of bowel-derived low-molecular weight molecules (< 20 kD) to access the central circulation. RESULTS: Results demonstrate a significant increase in systemic permeability to gut-derived peptides within 20 min after induction of hemorrhage (1.11 ± 0.19 vs 0.86 ± 0.07, P < 0.05) compared to control animals. Reperfusion resulted in a second, sustained increase in systemic permeability to gut-derived peptides in hemorrhaged animals compared to controls (1.2 ± 0.18 vs 0.97 ± 0.1, P < 0.05). Intravital microscopy of the mesentery also showed marked accumulation of fluorescent particles in the microcirculation of hemorrhaged animals compared to controls. These results were replicated using FITC dextrans 20 [10.85 ± 6.52 vs 3.38 ± 1.11 fluorescent intensity units (× 105, P < 0.05, hemorrhagic shock vs controls)], confirming that small bowel ischemia in response to experimental hemorrhagic shock results in marked and early increases in gut membrane permeability. CONCLUSION: Increased small bowel permeability in hemorrhagic shock may allow for systemic absorption of otherwise retained proteolytically-generated peptides, with consequent hemodynamic instability and remote organ failure. PMID:26557479

Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE

NASA Astrophysics Data System (ADS)

Hwang, Jeeseong; Giulian, Gary

2003-03-01

Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE Jeeseong Hwang, Jeffrey R. Krogmeier, Angela M. Bardo, Scott N. Goldie, Lori S. Goldner; Optical Technology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 Gary G. Giulian, Carl R. Merril; National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a popular method to separate proteins by their apparent molecular weight. However, it is a limited technique due, in part, to its low spatial resolution. In order to improve the resolution and to enhance the detection sensitivity of proteins separated by SDS-PAGE we are studying the detergent properties at the moving boundary of precast Tris-Tricine-Acetate cross-gradient gels using fluorescent cationic and pH indicating dyes. We have developed real-time full-field fluorescence polarization microscopy to monitor the dynamic fluorescence anisotropy from the cationic tetramethylindocarbocyanine dyes localized in the "extended stack", a concentrated detergent zone. We will present quantitative results of the fluorescence anisotropy. Our system is capable of analyzing local structures of the detergent molecules in the moving boundary of SDS-PAGE and the microenvironment(s) near the boundary. We will discuss the significance of these results and their potential role in enhanced protein separation.

Plasmonics Enhanced Smartphone Fluorescence Microscopy.

PubMed

Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

2017-05-18

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

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

Xie, Xiaoliang Sunney

Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly,more » even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.« less

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.

Spectral Demultiplexing in Holographic and Fluorescent On-chip Microscopy

NASA Astrophysics Data System (ADS)

Sencan, Ikbal; Coskun, Ahmet F.; Sikora, Uzair; Ozcan, Aydogan

2014-01-01

Lensfree on-chip imaging and sensing platforms provide compact and cost-effective designs for various telemedicine and lab-on-a-chip applications. In this work, we demonstrate computational solutions for some of the challenges associated with (i) the use of broadband, partially-coherent illumination sources for on-chip holographic imaging, and (ii) multicolor detection for lensfree fluorescent on-chip microscopy. Specifically, we introduce spectral demultiplexing approaches that aim to digitally narrow the spectral content of broadband illumination sources (such as wide-band light emitting diodes or even sunlight) to improve spatial resolution in holographic on-chip microscopy. We also demonstrate the application of such spectral demultiplexing approaches for wide-field imaging of multicolor fluorescent objects on a chip. These computational approaches can be used to replace e.g., thin-film interference filters, gratings or other optical components used for spectral multiplexing/demultiplexing, which can form a desirable solution for cost-effective and compact wide-field microscopy and sensing needs on a chip.

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

Uptake of Fluorescent Gentamicin by Peripheral Vestibular Cells after Systemic Administration

PubMed Central

Liu, Jianping; Kachelmeier, Allan; Dai, Chunfu; Li, Hongzhe; Steyger, Peter S.

2015-01-01

Objective In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. Results Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs’ sensory cells. Control vestibular tissues exposed to Dulbecco’s phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. Conclusions All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells. PMID:25793391

Aberrations and adaptive optics in super-resolution microscopy

PubMed Central

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-01-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips

NASA Astrophysics Data System (ADS)

Fetita, Catalin; Kirov, Boris; Jaramillo, Alfonso; Lefevre, Christophe

2012-03-01

With the accumulation of knowledge for the intimate molecular mechanisms governing the processes inside the living cells in the later years, the ability to characterize the performance of elementary genetic circuits and parts at the single-cell level is becoming of crucial importance. Biological science is arriving to the point where it can develop hypothesis for the action of each molecule participating in the biochemical reactions and need proper techniques to test those hypothesis. Microfluidics is emerging as the technology that combined with high-magnification microscopy will allow for the long-term single-cell level observation of bacterial physiology. In this study we design, build and characterize the gene dynamics of genetic circuits as one of the basic parts governing programmed cell behavior. We use E. coli as model organism and grow it in microfluidics chips, which we observe with epifluorescence microscopy. One of the most invaluable segments of this technology is the consequent image processing, since it allows for the automated analysis of vast amount of single-cell observation and the fast and easy derivation of conclusions based on that data. Specifically, we are interested in promoter activity as function of time. We expect it to be oscillatory and for that we use GFP (green fluorescent protein) as a reporter in our genetic circuits. In this paper, an automated framework for single-cell tracking in phase-contrast microscopy is developed, combining 2D segmentation of cell time frames and graph-based reconstruction of their spatiotemporal evolution with fast tracking of the associated fluorescence signal. The results obtained on the investigated biological database are presented and discussed.

Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO2 Nanotubes Using a Super-Oxidative Solution

PubMed Central

Beltrán-Partida, Ernesto; Moreno-Ulloa, Aldo; Valdez-Salas, Benjamín; Velasquillo, Cristina; Carrillo, Monica; Escamilla, Alan; Valdez, Ernesto; Villarreal, Francisco

2015-01-01

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V) is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO2 nanotube (NTs) layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX) and atomic force microscopy (AFM) were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials. PMID:28787976

Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO₂ Nanotubes Using a Super-Oxidative Solution.

PubMed

Beltrán-Partida, Ernesto; Moreno-Ulloa, Aldo; Valdez-Salas, Benjamín; Velasquillo, Cristina; Carrillo, Monica; Escamilla, Alan; Valdez, Ernesto; Villarreal, Francisco

2015-03-02

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V) is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO₂ nanotube (NTs) layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX) and atomic force microscopy (AFM) were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials.

Coherent nonlinear optical imaging: beyond fluorescence microscopy.

PubMed

Min, Wei; Freudiger, Christian W; Lu, Sijia; Xie, X Sunney

2011-01-01

The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.

Two Photon Intravital Microscopy of Lyme Borrelia in Mice.

PubMed

Belperron, Alexia A; Mao, Jialing; Bockenstedt, Linda K

2018-01-01

Two-photon intravital microscopy is a powerful tool that allows visualization of cells in intact tissues in a live animal in real time. In recent years, this advanced technology has been applied to understand pathogen-host interactions using fluorescently labeled bacteria. In particular, infectious fluorescent transformants of the Lyme disease spirochete Borrelia burgdorferi, an Ixodes tick-transmitted pathogen, have been imaged by two-photon intravital microscopy to study bacterial motility and interactions of the pathogen with feeding ticks and host tissues. Here, we describe the techniques and equipment used to image mammalian-adapted spirochetes in the skin of living mice in vivo and in joints ex vivo using two-photon intravital microscopy.

Guide to red fluorescent proteins and biosensors for flow cytometry.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-01-01

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Calibration of fluorescence resonance energy transfer in microscopy

DOEpatents

Youvan, Dougalas C.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Dilworth, Michael R.; Yang, Mary M.

2003-12-09

Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

Calibration of fluorescence resonance energy transfer in microscopy

DOEpatents

Youvan, Douglas C.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Dilworth, Michael R.; Yang, Mary M.

2002-09-24

Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

"Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching

NASA Astrophysics Data System (ADS)

Temirov, Jamshid; Werner, James H.; Goodwin, Peter M.; Bradbury, Andrew R. M.

2012-02-01

Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of fluorescent proteins displayed on T7 phage molecules.

Raman microscopy of bladder cancer cells expressing green fluorescent protein

NASA Astrophysics Data System (ADS)

Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

2016-11-01

Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

PubMed

Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

2013-02-01

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

PubMed

Chen, Robert; Rishi, Harneet S; Potapov, Vladimir; Yamada, Masaki R; Yeh, Vincent J; Chow, Thomas; Cheung, Celia L; Jones, Austin T; Johnson, Terry D; Keating, Amy E; DeLoache, William C; Dueber, John E

2015-11-20

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

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.

Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

NASA Astrophysics Data System (ADS)

Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

1995-04-01

We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using fluorescence in situ hybridization image is useful for the diagnosis of many other type of diseases, the system we have developed should find numerous applications for the diagnosis of disease states.

Decorating multi-walled carbon nanotubes with quantum dots for construction of multi-color fluorescent nanoprobes.

PubMed

Jia, Nengqin; Lian, Qiong; Tian, Zhong; Duan, Xin; Yin, Min; Jing, Lihong; Chen, Shouhui; Shen, Hebai; Gao, Mingyuan

2010-01-29

Novel multi-color fluorescent nanoprobes were prepared by electrostatically assembling differently sized CdTe quantum dots on polyethylenimine (PEI) functionalized multi-walled carbon nanotubes (MWNTs). The structural and optical properties of the nano-assemblies (MWNTs-PEI-CdTe) were characterized by transmission electron microscopy (TEM), electron diffraction spectra (EDS), Raman spectroscopy, confocal microscopy and photoluminescence spectroscopy (PL), respectively. Electrochemical impedance spectroscopy (EIS) was also applied to investigate the electrostatic assembling among oxidized MWNTs, PEI and CdTe. Furthermore, confocal fluorescence microscopy was used to monitor the nano-assemblies' delivery into tumor cells. It was found that the nano-assemblies exhibit efficient intracellular transporting and strong intracellular tracking. These properties would make this luminescent nano-assembly an excellent building block for the construction of intracellular nanoprobes, which could hold great promise for biomedical applications.

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…

Image correlation microscopy for uniform illumination.

PubMed

Gaborski, T R; Sealander, M N; Ehrenberg, M; Waugh, R E; McGrath, J L

2010-01-01

Image cross-correlation microscopy is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. Image cross-correlation microscopy has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy. In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy. Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning image cross-correlation microscopy, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function. Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function depends strongly on particle size and not particle shape. In this report, we establish the relationships between the spatial autocorrelation function feature size, temporal autocorrelation function characteristic time and the diffusion coefficient for uniform illumination image correlation microscopy using analytical, Monte Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate uniform illumination image correlation microscopy analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils.

High resolution multiple excitation spot optical microscopy

NASA Astrophysics Data System (ADS)

Dilipkumar, Shilpa; Mondal, Partha Pratim

2011-06-01

We propose fundamental improvements in three-dimensional (3D) resolution of multiple excitation spot optical microscopy. The excitation point spread function (PSF) is generated by two interfering counter-propagating depth-of-focus beams along the optical axis. Detection PSF is obtained by coherently interfering the emitted fluorescent light (collected by both the objectives) at the detector. System PSF shows upto 14-fold reduction in focal volume as compared to confocal, and almost 2-fold improvement in lateral resolution. Proposed PSF has the ability to simultaneously excite multiple 3D-spots of sub-femtoliter volume. Potential applications are in fluorescence microscopy and nanobioimaging.

Efficient blue conversion from a 1064 nm microchip laser in long photonic crystal fiber tapers for fluorescence microscopy.

PubMed

Kudlinski, A; Lelek, M; Barviau, B; Audry, L; Mussot, A

2010-08-02

Using a low-cost microchip laser and a long photonic crystal fiber taper, we report a supercontinuum source with a very efficient visible conversion, especially in the blue region (around 420 nm). About 30 % of the total average output power is located in the 350-600 nm band, which is of primary importance in a number of biophotonics applications such as flow cytometry or fluorescence imaging microscopy for instance. We successfully demonstrate the use of this visible-enhanced source for a three-color imaging of HeLa cells in wide-field microscopy.

Real-time fluorescence microscopy monitoring of porphyrin biodistribution

NASA Astrophysics Data System (ADS)

Kimel, Sol; Gottfried, Varda; Kunzi-Rapp, Karin; Akguen, Nermin; Schneckenburger, Herbert

1996-01-01

In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (Fmatrix) from the intravascular fluorescence intensity (Fintravascular), after transformation of the video frames into digital form. The differential fluorescence intensity, Fintravascular - Fmatrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

PubMed

Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

2015-01-01

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Clinical Nonlinear Laser Imaging of Human Skin: A Review

PubMed Central

Pavone, Francesco Saverio

2014-01-01

Nonlinear optical microscopy has the potential of being used in vivo as a noninvasive imaging modality for both epidermal and dermal imaging. This paper reviews the capabilities of nonlinear microscopy as a noninvasive high-resolution tool for clinical skin inspection. In particular, we show that two-photon fluorescence microscopy can be used as a diagnostic tool for characterizing epidermal layers by means of a morphological examination. Additional functional information on the metabolic state of cells can be provided by measuring the fluorescence decay of NADH. This approach allows differentiating epidermal layers having different structural and cytological features and has the potential of diagnosing pathologies in a very early stage. Regarding therapy follow-up, we demonstrate that nonlinear microscopy could be successfully used for monitoring the effect of a treatment. In particular, combined two-photon fluorescence and second-harmonic generation microscopy were used in vivo for monitoring collagen remodeling after microablative fractional laser resurfacing and for quantitatively monitoring psoriasis on the basis of the morphology of epidermal cells and dermal papillae. We believe that the described microscopic modalities could find in the near future a stable place in a clinical dermatological setting for quantitative diagnostic purposes and as a monitoring method for various treatments. PMID:25250337

CO2-switchable fluorescence of a dendritic polymer and its applications

NASA Astrophysics Data System (ADS)

Gao, Chunmei; Lü, Shaoyu; Liu, Mingzhu; Wu, Can; Xiong, Yun

2015-12-01

The synthesis and properties of CO2 responsive and fluorescent dendritic polymers, poly(amido amine)/Pluronic F127 (PAMAM/F127), are reported in this paper. The morphologies and sizes of PAMAM/F127 dendritic polymers were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). PAMAM/F127 dendritic polymers showed unimolecular micelle morphologies at low concentrations, and changed to multimolecular micelles at higher concentrations. Additionally, fluorescence spectra and confocal laser scanning microscopy images showed that PAMAM/F127 dendritic polymers exhibited a fluorescent enhancement response to the presence of CO2. Apart from that, the release behavior of PAMAM/F127 gels under simulated body fluids was investigated by choosing curcumin as the hydrophobic drug. The results indicated that PAMAM/F127 dendritic polymers can be used to improve the solubility of curcumin, and the drug released faster in the presence of CO2. Such CO2 responsive fluorescent dendritic polymers are potentially applicable in cellular imaging or drug controlled release.The synthesis and properties of CO2 responsive and fluorescent dendritic polymers, poly(amido amine)/Pluronic F127 (PAMAM/F127), are reported in this paper. The morphologies and sizes of PAMAM/F127 dendritic polymers were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). PAMAM/F127 dendritic polymers showed unimolecular micelle morphologies at low concentrations, and changed to multimolecular micelles at higher concentrations. Additionally, fluorescence spectra and confocal laser scanning microscopy images showed that PAMAM/F127 dendritic polymers exhibited a fluorescent enhancement response to the presence of CO2. Apart from that, the release behavior of PAMAM/F127 gels under simulated body fluids was investigated by choosing curcumin as the hydrophobic drug. The results indicated that PAMAM/F127 dendritic polymers can be used to improve the solubility of curcumin, and the drug released faster in the presence of CO2. Such CO2 responsive fluorescent dendritic polymers are potentially applicable in cellular imaging or drug controlled release. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06729d

Comparison of pre-processing techniques for fluorescence microscopy images of cells labeled for actin.

PubMed

Muralidhar, Gautam S; Channappayya, Sumohana S; Slater, John H; Blinka, Ellen M; Bovik, Alan C; Frey, Wolfgang; Markey, Mia K

2008-11-06

Automated analysis of fluorescence microscopy images of endothelial cells labeled for actin is important for quantifying changes in the actin cytoskeleton. The current manual approach is laborious and inefficient. The goal of our work is to develop automated image analysis methods, thereby increasing cell analysis throughput. In this study, we present preliminary results on comparing different algorithms for cell segmentation and image denoising.

Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues

NASA Astrophysics Data System (ADS)

Zheng, Wei

2014-11-01

Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.

Enhanced speed in fluorescence imaging using beat frequency multiplexing

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

2016-03-01

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

Development and Application of Chemical Probes for Vibrational Imaging by Stimulated Raman Scattering

NASA Astrophysics Data System (ADS)

Hu, Fanghao

During the last decade, Raman microscopy is experiencing rapid development and increasingly applied in biological and medical systems. Especially, stimulated Raman scattering (SRS) microscopy, which significantly improves the sensitivity of Raman scattering through stimulated emission, has allowed direct visualization of many species that are previously challenging with conventional fluorescence imaging. Compared to fluorescence, SRS imaging requires no label or small label on the target molecule, thus with minimal perturbation to the molecule of interest. Moreover, Raman scattering is free from complicated photophysical and photochemical processes such as photobleaching, and has intrinsically narrower linewidth than fluorescence emission. This allows multiplexed Raman imaging with minimal spectral crosstalk and excellent photo-stability. To achieve the full potential of Raman microscopy, vibrational probes have been developed for Raman imaging. Multiple Raman probes with a few atoms in size are applied in Raman imaging with high sensitivity and specificity. An overview of both fluorescence and Raman microscopy and their imaging probes is given in Chapter 1 with a brief discussion on the SRS theory. Built on the current progress of Raman microscopy and vibrational probes, I write on my research in the development of carbon-deuterium, alkyne and nitrile probes for visualizing choline metabolism (Chapter 2), glucose uptake activity (Chapter 3), complex brain metabolism (Chapter 4) and polymeric nanoparticles (Chapter 5) in live cells and tissues, as well as the development of polyyne-based vibrational probes for super-multiplexed imaging, barcoding and analysis (Chapter 6).

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.

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

PubMed Central

Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

2009-01-01

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

Mapping of Heavy Metal Ion Sorption to Cell-Extracellular Polymeric Substance-Mineral Aggregates by Using Metal-Selective Fluorescent Probes and Confocal Laser Scanning Microscopy

PubMed Central

Li, Jianli; Kappler, Andreas; Obst, Martin

2013-01-01

Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe3+, Cu2+, Zn2+, and Hg2+, illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems. PMID:23974141

Contrast Induced by a Static Magnetic Field for Improved Detection in Nanodiamond Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Singam, Shashi K. R.; Motylewski, Jaroslaw; Monaco, Antonina; Gjorgievska, Elena; Bourgeois, Emilie; Nesládek, Milos; Giugliano, Michele; Goovaerts, Etienne

2016-12-01

Diamond nanoparticles with negatively charged nitrogen-vacancy (NV) centers are highly efficient nonblinking emitters that exhibit spin-dependent intensity. An attractive application of these emitters is background-free fluorescence microscopy exploiting the fluorescence quenching induced either by resonant microwaves (RMWs) or by an applied static magnetic field (SMF). Here, we compare RMW- and SMF-induced contrast measurements over a wide range of optical excitation rates for fluorescent nanodiamonds (FNDs) and for NV centers shallowly buried under the (100)-oriented surface of a diamond single crystal (SC). Contrast levels are found to be systematically lower in the FNDs than in the SC. At low excitation rates, the RMW contrast initially rises to a maximum (up to 7% in FNDs and 13% in the SC) but then decreases steadily at higher intensities. Conversely, the SMF contrast increases from approximately 12% at low excitation rates to high values of 20% and 38% for the FNDs and SC, respectively. These observations are well described in a rate-equations model for the charged NV defect using parameters in good agreement with the literature. The SMF approach yields higher induced contrast in image collection under commonly applied optical excitation. Unlike the RMW method, there is no thermal load exerted on the aqueous media in biological samples in the SMF approach. We demonstrate imaging by SMF-induced contrast in neuronal cultures incorporating FNDs (i) in a setup for patch-clamp experiments in parallel with differential-interference-contrast microscopy, (ii) after a commonly used staining procedure as an illustration of the high selectivity against background fluorescence, and (iii) in a confocal fluorescence microscope in combination with bright-field microscopy.

Fluorescent microscopy and Ziehl-Neelsen staining of bronchoalveolar lavage, bronchial washings, bronchoscopic brushing and post bronchoscopic sputum along with cytological examination in cases of suspected tuberculosis.

PubMed

Bodal, Vijay Kumar; Bal, Manjit S; Bhagat, Sunita; Kishan, Jai; Brar, Rupinder K

2015-01-01

Ever since the discovery of Mycobacterium tuberculosis in 1882, many diagnostic methods have been developed. However "The gold standard" for the diagnosis of tuberculosis (TB) is still the demonstration of acid fast Bacilli (AFB) by microscopic examination of smear or bacteriological confirmation by culture method. In suspected 75 patients with active pulmonary TB, the materials obtained bronchoscopically, were bronchoalveolar lavage (BAL), bronchial brushings, bronchial washings and post bronchoscopic sputum. Four smears were made from each of the specimen. Fluorescent Staining, Ziehl-Neelsen (ZN), Pap and May Grunwald-Giemsa (MGG) stains were carried out for cytological examination. Fluorescent stain yielded maximum AFB positivity in all the methods, that is 36 (48%) in post fibre-optic bronchoscopy (FOB) sputum and 19 (25.33%) by fluorescence microscopy in both bronchial brushings and bronchial washings. Maximum yield of AFB with ZN staining 12 (16%) was equal to the post FOB sputum and bronchial brushings samples. It was followed by 6 cases (8%) in BAL and 4 (5.3%) in bronchial washings. The cytological examination was suggestive of TB in only 8 (10.66%) cases in bronchial washings and 6 (8%) cases in post FOB collection. It was equal in BAL and Bronchial brushings each that is 5 (6.67%). Bronchoscopy is a useful diagnostic tool and fluorescent microscopy is more sensitive than ZN and cytology. On X-ray examination, other diseases like malignancy or fungus can also mimick TB. So apart from ZN staining or fluorescence microscopy, Pap and MGG stain will be worthwhile to identify other microorganisms.

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers

PubMed Central

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-01-01

Background and Aims Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. Methods The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G0/G1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain–nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Key Results Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Conclusions Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family. PMID:27594649

A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs)

PubMed Central

Shachaf, Catherine M.; Elchuri, Sailaja V.; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N.; Mitchell, Dennis J.; Zhang, Jingwu; Swartz, Kenneth B.; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P.

2009-01-01

Background Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. Methodology/Principal Findings To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using “Composite Organic-Inorganic Nanoparticles” (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Conclusions/Significance Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells. PMID:19367337

Preparation of curcumin-loaded pluronic F127/chitosan nanoparticles for cancer therapy

NASA Astrophysics Data System (ADS)

Phuc Le, Thi Minh; Phuc Pham, Van; Lua Dang, Thi Minh; Huyen La, Thi; Hanh Le, Thi; Huan Le, Quang

2013-06-01

Nanoparticles (NPs) have been proven to be an effective delivery system with few side effects for anticancer drugs. In this study, curcumin-loaded NPs have been prepared by an ionic gelation method using chitosan (Chi) and pluronic®F-127 (PF) as carriers to deliver curcumin to the target cancer cells. Prepared NPs were characterized using Zetasizer, fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our results showed that the encapsulation efficiency of curcumin was approximately 50%. The average size of curcumin-loaded PF/Chi NPs was 150.9 nm, while the zeta potential was 5.09 mV. Cellular uptake of curcumin-loaded NPs into HEK293 cells was confirmed by fluorescence microscopy.

Resolution Limits of Nanoimprinted Patterns by Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Kubo, Shoichi; Tomioka, Tatsuya; Nakagawa, Masaru

2013-06-01

The authors investigated optical resolution limits to identify minimum distances between convex lines of fluorescent dye-doped nanoimprinted resist patterns by fluorescence microscopy. Fluorescent ultraviolet (UV)-curable resin and thermoplastic resin films were transformed into line-and-space patterns by UV nanoimprinting and thermal nanoimprinting, respectively. Fluorescence immersion observation needed an immersion medium immiscible to the resist films, and an ionic liquid of triisobutyl methylphosphonium tosylate was appropriate for soluble thermoplastic polystyrene patterns. Observation with various numerical aperture (NA) values and two detection wavelength ranges showed that the resolution limits were smaller than the values estimated by the Sparrow criterion. The space width to identify line patterns became narrower as the line width increased. The space width of 100 nm was demonstrated to be sufficient to resolve 300-nm-wide lines in the detection wavelength range of 575-625 nm using an objective lens of NA= 1.40.

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

PubMed

Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

2012-01-01

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

A small molecular pH-dependent fluorescent probe for cancer cell imaging in living cell.

PubMed

Ma, Junbao; Li, Wenqi; Li, Juanjuan; Shi, Rongguang; Yin, Gui; Wang, Ruiyong

2018-05-15

A novel pH-dependent two-photon fluorescent molecular probe ABMP has been prepared based on the fluorophore of 2, 4, 6-trisubstituted pyridine. The probe has an absorption wavelength at 354 nm and corresponding emission wavelength at 475 nm with the working pH range from 2.20 to 7.00, especially owning a good liner response from pH = 2.40 to pH = 4.00. ABMP also has excellent reversibility, photostability and selectivity which promotes its ability in analytical application. The probe can be excited with a two-photon fluorescence microscopy and the fluorescence cell imaging indicated that the probe can distinguish Hela cancer cells out of normal cells with a two-photon fluorescence microscopy which suggested its potential application in tumor cell detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

NASA Astrophysics Data System (ADS)

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

PubMed

Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

2016-08-01

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular engineering of two-photon fluorescent probes for bioimaging applications

NASA Astrophysics Data System (ADS)

Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

2017-03-01

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

Porphyrin involvement in redshift fluorescence in dentin decay

NASA Astrophysics Data System (ADS)

Slimani, A.; Panayotov, I.; Levallois, B.; Cloitre, T.; Gergely, C.; Bec, N.; Larroque, C.; Tassery, H.; Cuisinier, F.

2014-05-01

The aim of this study was to evaluate the porphyrin involvement in the red fluorescence observed in dental caries with Soprolife® light-induced fluorescence camera in treatments mode (SOPRO, ACTEON Group, La Ciotat, France) and Vistacam® camera (DÜRR DENTAL AG, Bietigheim-Bissingen, Germany). The International Caries Detection and Assessment System (ICDAS) was used to rand the samples. Human teeth cross-sections, ranked from ICDAS score 0 to 6, were examined by epi-fluorescence microscopy and Confocal Raman microscopy. Comparable studies were done with Protoporphyrin IX, Porphyrin I and Pentosidine solutions. An RGB analysis of Soprolife® images was performed using ImageJ Software (1.46r, National Institutes of Health, USA). Fluorescence spectroscopy and MicroRaman spectroscopy revealed the presence of Protoporphyrin IX, in carious enamel, dentin and dental plaque. However, the presence of porphyrin I and pentosidine cannot be excluded. The results indicated that not only porphyrin were implicated in the red fluorescence, Advanced Glygation Endproducts (AGEs) of the Maillard reaction also contributed to this phenomenon.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

PubMed Central

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-01-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy. PMID:28300146

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

PubMed Central

van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

Highly fluorescent peptide nanoribbon impregnated with Sn-porphyrin as a potent DNA sensor.

PubMed

Parayil, Sreenivasan Koliyat; Lee, Jooran; Yoon, Minjoong

2013-05-01

Highly fluorescent and thermo-stable peptide nanoribbons (PNRs) were fabricated by solvothermal self-assembly of a single peptide (D,D-diphenyl alanine peptides) with Sn-porphyrin (trans-dihydroxo[5,10,15,20-tetrakis(p-tolyl)porphyrinato] Sn(IV) (SnTTP(OH)2)). The structural characterization of the as-prepared nanoribbons was performed by transmitting electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM), FT-IR and Raman spectroscopy, indicating that the lipophilic Sn-porphyrins are impregnated into the porous surface formed in the process of nanoribbon formation through intermolecular hydrogen bonding of the peptide main chains. Consequently the Sn-porphyrin-impregnated peptide nanoribbons (Sn-porphyrin-PNRs) exhibited typical UV-visible absorption spectrum of the monomer porphyrin with a red shifted Q-band, and their fluorescence quantum yield was observed to be enhanced compared to that of free Sn-porphyrin. Interestingly the fluorescence intensity and lifetimes of Sn-porphyrin-PNRs were selectively affected upon interaction with nucleotide base sequences of DNA while those of free Sn-porphyrins were not affected by binding with any of the DNA studied, indicating that DNA-induced changes in the fluorescence properties of Sn-porphyrin-PNRs are due to interaction between DNA and the PNR scaffold. These results imply that Sn-porphyrin-PNR will be useful as a potent fluorescent protein analogue and as a biocompatible DNA sensor.

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

PubMed Central

Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

2013-01-01

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

Non-Cytotoxic Quantum Dot–Chitosan Nanogel Biosensing Probe for Potential Cancer Targeting Agent

PubMed Central

Maxwell, Tyler; Banu, Tahmina; Price, Edward; Tharkur, Jeremy; Campos, Maria Gabriela Nogueira; Gesquiere, Andre; Santra, Swadeshmukul

2015-01-01

Quantum dot (Qdot) biosensors have consistently provided valuable information to researchers about cellular activity due to their unique fluorescent properties. Many of the most popularly used Qdots contain cadmium, posing the risk of toxicity that could negate their attractive optical properties. The design of a non-cytotoxic probe usually involves multiple components and a complex synthesis process. In this paper, the design and synthesis of a non-cytotoxic Qdot-chitosan nanogel composite using straight-forward cyanogen bromide (CNBr) coupling is reported. The probe was characterized by spectroscopy (UV-Vis, fluorescence), microscopy (Fluorescence, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. This activatable (“OFF”/“ON”) probe contains a core–shell Qdot (CdS:Mn/ZnS) capped with dopamine, which acts as a fluorescence quencher and a model drug. Dopamine capped “OFF” Qdots can undergo ligand exchange with intercellular glutathione, which turns the Qdots “ON” to restore fluorescence. These Qdots were then coated with chitosan (natural biocompatible polymer) functionalized with folic acid (targeting motif) and Fluorescein Isothiocyanate (FITC; fluorescent dye). To demonstrate cancer cell targetability, the interaction of the probe with cells that express different folate receptor levels was analyzed, and the cytotoxicity of the probe was evaluated on these cells and was shown to be nontoxic even at concentrations as high as 100 mg/L. PMID:28347126

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

PubMed Central

Klauss, André; König, Marcelle; Hille, Carsten

2015-01-01

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating. PMID:26091552

Lytic resistance of fibrin containing red blood cells

PubMed Central

Wohner, Nikolett; Sótonyi, Péter; Machovich, Raymund; Szabó, László; Tenekedjiev, Kiril; Silva, Marta M.C.G.; Longstaff, Colin; Kolev, Krasimir

2012-01-01

Objective Arterial thrombi contain variable amounts of red blood cell (RBC), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. Here we evaluate the modulator role of RBCs in the lytic susceptibility of fibrin. Methods and Results If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 nm to 96 nm at 40 %(v/v) RBC, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBC prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6 %, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein (GFP)-labeled tPA and orange fluorescent fibrin showed that 20-40 %(v/v) RBC significantly slowed down the dissolution of the clots. tPA-GFP did not accumulate on the surface of fibrin containing RBC at any cell count above 10 %. The presence of RBC in the clot suppressed the tPA-induced plasminogen activation resulting in a 45 % less plasmin generated after 30 min activation at 40 %(v/v) RBC. Conclusion RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions. PMID:21737785

Morphology and Histochemistry of the Glandular Trichomes of Lippia scaberrima (Verbenaceae)

PubMed Central

Combrinck, S.; Du Plooy, G. W.; McCrindle, R. I.; Botha, B. M.

2007-01-01

Background and Aims Lippia scaberrima, an aromatic indigenous South African plant, with medicinal application, potentially has economic value. The production of essential oil from this plant has not been optimized, and this study of the chemico-morphological characteristics was aimed at determining the location of oil production within the plant. Furthermore, the locality of other secondary metabolites important in medicinal applications needed to be ascertained. This information would be useful in deciding the protocol required for isolation of such compounds. Methods The morphology of the glandular trichomes was investigated using a combination of scanning electron and light microscopy. Concurrently, the chemical content was studied by applying various chemical reagents and fluorescence microscopy. Key Results Three types of trichomes were distinguished on the material investigated. Large, bulbous peltate glands containing compounds of terpenoid nature are probably the main site of essential oil accumulation. Small glands were found to be both peltate and capitate and fluorescent stain indicated the possible presence of phenolic compounds. The third type was a slender tapered seta with an ornamented surface and uniseriate base, and evidently secretory in nature. Conclusions This study linking the chemical content and morphology of the glandular trichomes of L. scaberrima has contributed to the knowledge and understanding of secretory structures of Lippia spp. in general. PMID:17468110

Richardson-Lucy/maximum likelihood image restoration algorithm for fluorescence microscopy: further testing.

PubMed

Holmes, T J; Liu, Y H

1989-11-15

A maximum likelihood based iterative algorithm adapted from nuclear medicine imaging for noncoherent optical imaging was presented in a previous publication with some initial computer-simulation testing. This algorithm is identical in form to that previously derived in a different way by W. H. Richardson "Bayesian-Based Iterative Method of Image Restoration," J. Opt. Soc. Am. 62, 55-59 (1972) and L. B. Lucy "An Iterative Technique for the Rectification of Observed Distributions," Astron. J. 79, 745-765 (1974). Foreseen applications include superresolution and 3-D fluorescence microscopy. This paper presents further simulation testing of this algorithm and a preliminary experiment with a defocused camera. The simulations show quantified resolution improvement as a function of iteration number, and they show qualitatively the trend in limitations on restored resolution when noise is present in the data. Also shown are results of a simulation in restoring missing-cone information for 3-D imaging. Conclusions are in support of the feasibility of using these methods with real systems, while computational cost and timing estimates indicate that it should be realistic to implement these methods. Itis suggested in the Appendix that future extensions to the maximum likelihood based derivation of this algorithm will address some of the limitations that are experienced with the nonextended form of the algorithm presented here.

Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

NASA Astrophysics Data System (ADS)

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2010-03-01

In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

PubMed

Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

2015-11-02

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

Application of spectroscopy and super-resolution microscopy: Excited state

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

Bhattacharjee, Ujjal

Photophysics of inorganic materials and organic molecules in complex systems have been extensively studied with absorption and emission spectroscopy.1-4 Steady-state and time-resolved fluorescence studies are commonly carried out to characterize excited-state properties of fluorophores. Although steady-state fluorescence measurements are widely used for analytical applications, time-resolved fluorescence measurements provide more detailed information about excited-state properties and the environment in the vicinity of the fluorophore. Many photophysical processes, such as photoinduced electron transfer (PET), rotational reorientation, solvent relaxation, and energy transfer, occur on a nanosecond (10 -9 s) timescale, thus affecting the lifetime of the fluorophores. Moreover, time-resolved microscopy methods, such asmore » lifetimeimaging, combine the benefits of the microscopic measurement and information-rich, timeresolved data. Thus, time-resolved fluorescence spectroscopy combined with microscopy can be used to quantify these processes and to obtain a deeper understanding of the chemical surroundings of the fluorophore in a small area under investigation. This thesis discusses various photophysical and super-resolution microscopic studies of organic and inorganic materials, which have been outlined below.« less

A novel fluorescence microscopy approach to estimate quality loss of stored fruit fillings as a result of browning.

PubMed

Cropotova, Janna; Tylewicz, Urszula; Cocci, Emiliano; Romani, Santina; Dalla Rosa, Marco

2016-03-01

The aim of the present study was to estimate the quality deterioration of apple fillings during storage. Moreover, a potentiality of novel time-saving and non-invasive method based on fluorescence microscopy for prompt ascertainment of non-enzymatic browning initiation in fruit fillings was investigated. Apple filling samples were obtained by mixing different quantities of fruit and stabilizing agents (inulin, pectin and gellan gum), thermally processed and stored for 6-month. The preservation of antioxidant capacity (determined by DPPH method) in apple fillings was indirectly correlated with decrease in total polyphenols content that varied from 34±22 to 56±17% and concomitant accumulation of 5-hydroxymethylfurfural (HMF), ranging from 3.4±0.1 to 8±1mg/kg in comparison to initial apple puree values. The mean intensity of the fluorescence emission spectra of apple filling samples and initial apple puree was highly correlated (R(2)>0.95) with the HMF content, showing a good potentiality of fluorescence microscopy method to estimate non-enzymatic browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

Correction for photobleaching in dynamic fluorescence microscopy: application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery

NASA Astrophysics Data System (ADS)

Derieppe, M.; Bos, C.; de Greef, M.; Moonen, C.; de Senneville, B. Denis

2016-01-01

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally. Photobleaching rates up to 5.0 10-3 s-1 were measured when applying power densities up to 0.2 W.cm-2. By applying the three-compartment model, the model drug uptake rate of 6.0 10-3 s-1 was measured independent of the applied laser power. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization.

Using DNA origami nanorulers as traceable distance measurement standards and nanoscopic benchmark structures.

PubMed

Raab, Mario; Jusuk, Ija; Molle, Julia; Buhr, Egbert; Bodermann, Bernd; Bergmann, Detlef; Bosse, Harald; Tinnefeld, Philip

2018-01-29

In recent years, DNA origami nanorulers for superresolution (SR) fluorescence microscopy have been developed from fundamental proof-of-principle experiments to commercially available test structures. The self-assembled nanostructures allow placing a defined number of fluorescent dye molecules in defined geometries in the nanometer range. Besides the unprecedented control over matter on the nanoscale, robust DNA origami nanorulers are reproducibly obtained in high yields. The distances between their fluorescent marks can be easily analysed yielding intermark distance histograms from many identical structures. Thus, DNA origami nanorulers have become excellent reference and training structures for superresolution microscopy. In this work, we go one step further and develop a calibration process for the measured distances between the fluorescent marks on DNA origami nanorulers. The superresolution technique DNA-PAINT is used to achieve nanometrological traceability of nanoruler distances following the guide to the expression of uncertainty in measurement (GUM). We further show two examples how these nanorulers are used to evaluate the performance of TIRF microscopes that are capable of single-molecule localization microscopy (SMLM).

Interrogating Surface Functional Group Heterogeneity of Activated Thermoplastics Using Super-Resolution Fluorescence Microscopy.

PubMed

ONeil, Colleen E; Jackson, Joshua M; Shim, Sang-Hee; Soper, Steven A

2016-04-05

We present a novel approach for characterizing surfaces utilizing super-resolution fluorescence microscopy with subdiffraction limit spatial resolution. Thermoplastic surfaces were activated by UV/O3 or O2 plasma treatment under various conditions to generate pendant surface-confined carboxylic acids (-COOH). These surface functional groups were then labeled with a photoswitchable dye and interrogated using single-molecule, localization-based, super-resolution fluorescence microscopy to elucidate the surface heterogeneity of these functional groups across the activated surface. Data indicated nonuniform distributions of these functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of -COOH groups secured from super-resolution imaging were used to simulate nonuniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process.

Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems.

PubMed

Yan, Yuling; Marriott, M Emma; Petchprayoon, Chutima; Marriott, Gerard

2011-02-01

Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

Two-Photon Fluorescence Microscope for Microgravity Research

NASA Technical Reports Server (NTRS)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2005-01-01

A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the longer-wavelength excitation light and passes the shorter-wavelength fluorescence light. Also, the confocal pinhole has been removed to increase the signal strength. The laser beam is scanned by a twoperpendicular- axis pair of galvanometer mirrors through a pupil transfer lens into the side port of an inverted microscope. Finally, the beam is focused by a 63-magnification, 1.3-numerical- aperture oil-immersion objective lens onto a specimen. The pupil transfer lens serves to match the intermediate image planes of the scanning head and the microscope, and its location is critical. In order to maximize the quality of the image, (that is, the point spread function of the objective lens for all scan positions), the entire system was modeled in optical-design software, and the various free design parameters (the parameters of the spatial-filter components as well as the separations of all of the system components) were determined through an iterative optimization process. A modular design was chosen to facilitate access to the optical train for future fluorescence correlation spectroscopy and fluorescence-lifetime experiments.

Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy.

PubMed

Mandavilli, Bhaskar S; Janes, Michael S

2010-07-01

Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell-permeant, thiol-reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection modalities. While this probe has been validated in flow cytometry and microplate fluorimetry assays, the following method will describe details on the use of the ThiolTracker Violet dye in traditional fluorescence microscopy, as well as high-content imaging and analysis.

Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

PubMed Central

Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, Rainer

2016-01-01

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles. PMID:27783656

A user's guide to localization-based super-resolution fluorescence imaging.

PubMed

Dempsey, Graham T

2013-01-01

Advances in far-field fluorescence microscopy over the past decade have led to the development of super-resolution imaging techniques that provide more than an order of magnitude improvement in spatial resolution compared to conventional light microscopy. One such approach, called Stochastic Optical Reconstruction Microscopy (STORM) uses the sequential, nanometer-scale localization of individual fluorophores to reconstruct a high-resolution image of a structure of interest. This is an attractive method for biological investigation at the nanoscale due to its relative simplicity, both conceptually and practically in the laboratory. Like most research tools, however, the devil is in the details. The aim of this chapter is to serve as a guide for applying STORM to the study of biological samples. This chapter will discuss considerations for choosing a photoswitchable fluorescent probe, preparing a sample, selecting hardware for data acquisition, and collecting and analyzing data for image reconstruction. Copyright © 2013 Elsevier Inc. All rights reserved.

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

PubMed

Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

2015-01-27

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

NASA Astrophysics Data System (ADS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

PubMed Central

Gulati, Srishti; Cao, Vania Y.; Otte, Stephani

2017-01-01

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms. PMID:28654056

Reconstitution radicicol containing apolipoprotein B lipoparticle and tracing its cell uptake process by super resolution fluorescent microscopy.

NASA Astrophysics Data System (ADS)

Lin, Chung Ching; Lin, Po-Yen; Chang, Chia-Ching

Apolipoprotein B (apoB) is the only protein of LDL. LDL delivers cholesterol, triacylglycerides and lipids to the target cells. Reconstitute apoB lipoparticle (rABL) will be an idea drug delivery vehicle for hydrophobic and amphiphilic materials delivery. It is challenged to renature ApoB into its functional state from denatured state. By using modified bile salt and radicicol (Rad) added over-critical refolding process, apoB can be restored into its native like state. The intrinsic fluorescence of apoB increased during the refolding process. Moreover, radicicol (Rad) molecules have been encapsulated into reconstitute rABL (Rad@rABL). To investigate the cell uptake mechanism of Rad@rABL, a super resolution ground state depletion (GSD) microscopy is used in this research. Fluorescence labeled Rad@rABL can be traced within the tumor cell. Key words: LDL, radicicol, protein refolding, super resolution microscopy.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy.

PubMed

Gu, Min; Bird, Damian

2003-05-01

The three-dimensional optical transfer function is derived for analyzing the imaging performance in fiber-optical two-photon fluorescence microscopy. Two types of fiber-optical geometry are considered: The first involves a single-mode fiber for delivering a laser beam for illumination, and the second is based on the use of a single-mode fiber coupler for both illumination delivery and signal collection. It is found that in the former case the transverse and axial cutoff spatial frequencies of the three-dimensional optical transfer function are the same as those in conventional two-photon fluorescence microscopy without the use of a pinhole.However, the transverse and axial cutoff spatial frequencies in the latter case are 1.7 times as large as those in the former case. Accordingly, this feature leads to an enhanced optical sectioning effect when a fiber coupler is used, which is consistent with our recent experimental observation.

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

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.

Fundus Autofluorescence and Photoreceptor Cell Rosettes in Mouse Models

PubMed Central

Flynn, Erin; Ueda, Keiko; Auran, Emily; Sullivan, Jack M.; Sparrow, Janet R.

2014-01-01

Purpose. This study was conducted to study correlations among fundus autofluorescence (AF), RPE lipofuscin accumulation, and photoreceptor cell degeneration and to investigate the structural basis of fundus AF spots. Methods. Fundus AF images (55° lens; 488-nm excitation) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired in pigmented Rdh8−/−/Abca4−/− mice (ages 1–9 months) with a confocal scanning laser ophthalmoscope (cSLO). For quantitative fundus AF (qAF), gray levels (GLs) were calibrated to an internal fluorescence reference. Retinal bisretinoids were measured by quantitative HPLC. Histometric analysis of outer nuclear layer (ONL) thicknesses was performed, and cryostat sections of retina were examined by fluorescence microscopy. Results. Quantified A2E and qAF intensities increased until age 4 months in the Rdh8−/−/Abca4−/− mice. The A2E levels declined after 4 months of age, but qAF intensity values continued to rise. The decline in A2E levels in the Rdh8−/−/Abca4−/− mice paralleled reduced photoreceptor cell viability as reflected in ONL thinning. Hyperautofluorescent puncta in fundus AF images corresponded to photoreceptor cell rosettes in SD-OCT images and histological sections stained with hematoxylin and eosin. The inner segment/outer segment–containing core of the rosette emitted an autofluorescence detected by fluorescence microscopy. Conclusions. When neural retina is disordered, AF from photoreceptor cells can contribute to noninvasive fundus AF images. Hyperautofluorescent puncta in fundus AF images are attributable, in at least some cases, to photoreceptor cell rosettes. PMID:25015357

High resolution 3D imaging of synchrotron generated microbeams

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

Gagliardi, Frank M., E-mail: frank.gagliardi@wbrc.org.au; Cornelius, Iwan; Blencowe, Anton

2015-12-15

Purpose: Microbeam radiation therapy (MRT) techniques are under investigation at synchrotrons worldwide. Favourable outcomes from animal and cell culture studies have proven the efficacy of MRT. The aim of MRT researchers currently is to progress to human clinical trials in the near future. The purpose of this study was to demonstrate the high resolution and 3D imaging of synchrotron generated microbeams in PRESAGE® dosimeters using laser fluorescence confocal microscopy. Methods: Water equivalent PRESAGE® dosimeters were fabricated and irradiated with microbeams on the Imaging and Medical Beamline at the Australian Synchrotron. Microbeam arrays comprised of microbeams 25–50 μm wide with 200more » or 400 μm peak-to-peak spacing were delivered as single, cross-fire, multidirectional, and interspersed arrays. Imaging of the dosimeters was performed using a NIKON A1 laser fluorescence confocal microscope. Results: The spatial fractionation of the MRT beams was clearly visible in 2D and up to 9 mm in depth. Individual microbeams were easily resolved with the full width at half maximum of microbeams measured on images with resolutions of as low as 0.09 μm/pixel. Profiles obtained demonstrated the change of the peak-to-valley dose ratio for interspersed MRT microbeam arrays and subtle variations in the sample positioning by the sample stage goniometer were measured. Conclusions: Laser fluorescence confocal microscopy of MRT irradiated PRESAGE® dosimeters has been validated in this study as a high resolution imaging tool for the independent spatial and geometrical verification of MRT beam delivery.« less

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.

Fluorescence imaging of reactive oxygen species by confocal laser scanning microscopy for track analysis of synchrotron X-ray photoelectric nanoradiator dose: X-ray pump-optical probe.

PubMed

Jeon, Jae Kun; Han, Sung Mi; Kim, Jong Ki

2016-09-01

Bursts of emissions of low-energy electrons, including interatomic Coulomb decay electrons and Auger electrons (0-1000 eV), as well as X-ray fluorescence produced by irradiation of large-Z element nanoparticles by either X-ray photons or high-energy ion beams, is referred to as the nanoradiator effect. In therapeutic applications, this effect can damage pathological tissues that selectively take up the nanoparticles. Herein, a new nanoradiator dosimetry method is presented that uses probes for reactive oxygen species (ROS) incorporated into three-dimensional gels, on which macrophages containing iron oxide nanoparticles (IONs) are attached. This method, together with site-specific irradiation of the intracellular nanoparticles from a microbeam of polychromatic synchrotron X-rays (5-14 keV), measures the range and distribution of OH radicals produced by X-ray emission or superoxide anions ({\\rm{O}}_2^-) produced by low-energy electrons. The measurements are based on confocal laser scanning of the fluorescence of the hydroxyl radical probe 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) or the superoxide probe hydroethidine-dihydroethidium (DHE) that was oxidized by each ROS, enabling tracking of the radiation dose emitted by the nanoradiator. In the range 70 µm below the irradiated cell, ^\\bullet{\\rm{OH}} radicals derived mostly from either incident X-ray or X-ray fluorescence of ION nanoradiators are distributed along the line of depth direction in ROS gel. In contrast, {\\rm{O}}_2^- derived from secondary electron or low-energy electron emission by ION nanoradiators are scattered over the ROS gel. ROS fluorescence due to the ION nanoradiators was observed continuously to a depth of 1.5 mm for both oxidized APF and oxidized DHE with relatively large intensity compared with the fluorescence caused by the ROS produced solely by incident primary X-rays, which was limited to a depth of 600 µm, suggesting dose enhancement as well as more penetration by nanoradiators. In conclusion, the combined use of a synchrotron X-ray microbeam-irradiated three-dimensional ROS gel and confocal laser scanning fluorescence microscopy provides a simple dosimetry method for track analysis of X-ray photoelectric nanoradiator radiation, suggesting extensive cellular damage with dose-enhancement beyond a single cell containing IONs.

Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging†

PubMed Central

Digman, Michelle A.; Gratton, Enrico; Storti, Barbara; Beltram, Fabio

2013-01-01

A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy. PMID:22517076

Functionality-Oriented Derivatization of Naphthalene Diimide: A Molecular Gel Strategy-Based Fluorescent Film for Aniline Vapor Detection.

PubMed

Fan, Jiayun; Chang, Xingmao; He, Meixia; Shang, Congdi; Wang, Gang; Yin, Shiwei; Peng, Haonan; Fang, Yu

2016-07-20

Modification of naphthalene diimide (NDI) resulted in a photochemically stable, fluorescent 3,4,5-tris(dodecyloxy)benzamide derivative of NDI (TDBNDI), and introduction of the long alkyl chains endowed the compound with good compatibility with commonly found organic solvents and in particular superior self-assembly in the solution state. Further studies revealed that TDBNDI forms gels with nine of the 18 solvents tested at a concentration of 2.0% (w/v), and the critical gelation concentrations of five of the eight gels are lower than 1.0% (w/v), indicating the high efficiency of the compound as a low-molecular mass gelator (LMMG). Transmission electron microscopy, scanning electron microscopy, and confocal laser scanning microscopy studies revealed the networked fibrillar structure of the TDBNDI/methylcyclohexane (MCH) gel. On the basis of these findings, a fluorescent film was developed via simple spin-coating of the TDBNDI/MCH gel on a glass substrate surface. Fluorescence behavior and sensing performance studies demonstrated that this film is photochemically stable, and sensitive and selective to the presence of aniline vapor. Notably, the response is instantaneous, and the sensing process is fully and quickly reversible. This case study demonstrates that derivatization of photochemically stable fluorophores into LMMGs is a good strategy for developing high-performance fluorescent sensing films.

Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands.

PubMed

Magnusson, Karin; Simon, Rozalyn; Sjölander, Daniel; Sigurdson, Christina J; Hammarström, Per; Nilsson, K Peter R

2014-01-01

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.

Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands

PubMed Central

Magnusson, Karin; Simon, Rozalyn; Sjölander, Daniel; Sigurdson, Christina J; Hammarström, Per; Nilsson, K Peter R

2014-01-01

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies. PMID:25495506

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

PubMed

Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

2016-04-01

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

NASA Astrophysics Data System (ADS)

Miao, Qin; Rahn, J. Richard; Tourovskaia, Anna; Meyer, Michael G.; Neumann, Thomas; Nelson, Alan C.; Seibel, Eric J.

2009-11-01

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 μm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

Potential of Fluorescence Imaging Techniques To Monitor Mutagenic PAH Uptake by Microalga

PubMed Central

2015-01-01

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold’s basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that BaP does not aggregate inside Chlorella sp. (average brightness = 5.330), while it aggregates in the supernatant. In Chlorella grown in sediments spiked with BaP, in 12 h the BaP uptake could be visualized using fluorescence microscopy. PMID:25020149

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform

PubMed Central

Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G.; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C.; Kummel, Andrew C.

2015-01-01

Abstract. A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N-hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg, respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

PubMed Central

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

DOE PAGES

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

2014-10-31

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

NASA Astrophysics Data System (ADS)

Gramaccioni, Chiara; Yang, Yang; Procopio, Alessandra; Pacureanu, Alexandra; Bohic, Sylvain; Malucelli, Emil; Iotti, Stefano; Farruggia, Giovanna; Bukreeva, Inna; Notargiacomo, Andrea; Fratini, Michela; Valenti, Piera; Rosa, Luigi; Berlutti, Francesca; Cloetens, Peter; Lagomarsino, Stefano

2018-01-01

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

Bacterial cell identification in differential interference contrast microscopy images.

PubMed

Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

2013-04-23

Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

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.

Stochastic Optical Reconstruction Microscopy (STORM).

PubMed

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Super resolution imaging of HER2 gene amplification

NASA Astrophysics Data System (ADS)

Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

2016-02-01

HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

Role of 5-ALA in improving extent of tumour resection in patients with Glioblastoma Multiforme.

PubMed

Waqas, Muhammad; Khan, Inamullah; Shamim, Muhammad Shahzad

2017-10-01

Goal of surgery for patients with Glioblastoma Multiforme (GBM) is gross total resection with no new neurological deficits. Surgical resection is often restricted due the difficulty in differentiating the tumour from surrounding normal brain using either naked eye, or standard intra-operative white light microscopy. GBM uptakes orally administered 5-ALA becomes fluorescent when viewed by a special light, and this property has been used to improve intra-operative tumour identification. This technique should therefore allow better extent of tumour resection. The hypothesis has been tested through several studies and even though most studies are of low quality, they strongly favour the use of 5- ALA in improving the extent of resection when compared to white light microscopy. A systematic review on the topic had a similar conclusion. Few studies have also hinted on a high false negative rate with the use of this technique..

Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens

PubMed Central

Elson, D S; Jo, J A

2007-01-01

We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues. PMID:19503759

Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia

PubMed Central

Frost, Emily; Dixon, Mellisa; Ollosson, Sarah; Kilpin, Kate; Patel, Mitali; Scully, Juliet; Rogers, Andrew V.; Mitchison, Hannah M.; Bush, Andrew; Hogg, Claire

2017-01-01

Rationale: The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the United Kingdom consists of assessing ciliary function by high-speed microscopy and ultrastructure by election microscopy, but equipment and expertise is not widely available internationally. The identification of biallelic disease-causing mutations is also diagnostic, but many disease-causing genes are unknown, and testing is not widely available outside the United States. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. Objectives: To determine utility of a panel of six fluorescent labeled antibodies as a diagnostic tool for PCD. Methods: The study used immunofluorescent labeling of nasal brushings from a discovery cohort of 35 patients diagnosed with PCD by ciliary ultrastructure, and a diagnostic accuracy cohort of 386 patients referred with symptoms suggestive of disease. The results were compared with diagnostic outcome. Measurements and Main Results: Immunofluorescence correctly identified mislocalized or absent staining in 100% of the discovery cohort. In the diagnostic cohort immunofluorescence successfully identified 22 of 25 patients with PCD and normal staining in all 252 in whom PCD was considered highly unlikely. In addition, immunofluorescence provided a result in 55% (39) of cases that were previously inconclusive. Immunofluorescence results were available within 14 days, costing $187 per sample compared with electron microscopy (27 days; cost $1,452). Conclusions: Immunofluorescence is a highly specific diagnostic test for PCD, and it improves the speed and availability of diagnostic testing. However, sensitivity is limited and immunofluorescence is not suitable as a stand-alone test. PMID:28199173

[Regression of glomerulosclerosis in a girl with C3 nephropathy].

PubMed

Jarmoliński, Tomasz; Borucka, Barbara; Wozniak, Aldona

2005-01-01

Regression of glomerular sclerosis was documented in experimental models as a result of RAA system blocking and/or cyclosporin A (CsA) treatment. Here we present a case of a girl suffering from nephrotic syndrome (NS) in whom unusual healing of glomerular changes appeared. First time the girl was admitted to our department at the age of 4 year with 2 years history of steroid-dependent NS. Kidney biopsy studied in light microscopy revealed 11 glomeruli among which 5 were totally sclerosed and the others showed mesangial proliferation and segmental mesangial matrix expansion. Diffuse interstitial mononuclear cells infiltration was also visible. In fluorescence microscopy only granular C3 and fibrinogen deposits were seen. C3 nephropathy was diagnosed and cyclophosphamide therapy started. As no effects appeared, CsA was introduced together with angiotensin converting enzyme inhibitor (ACEI) enalapril for two years without any relapse during treatment. Prednisone was gradually reduced and finally stopped after a year. On the second biopsy performed to assess CsA nephrotoxicity among 16 glomeruli evaluated in light microscopy only 1 was sclerosed, while the others presented just mild mesangial proliferation. No interstitial changes were found. Fluorescence showed IgA and IgM added to glomerular C3 deposits. CsA was then stopped and after 4 months relapses and steroid-dependency appeared again, so CsA was reintroduced. Actually the girl is 8-year-old, remission of NS has been observed for six months. As a conclusion we would like to suggest that treatment with CsA and ACEI may cause glomerular healing even in a case of advanced glomerulosclerosis.

Epifluorescence Intravital Microscopy of Murine Corneal Dendritic Cells

PubMed Central

Rosenbaum, James T.; Planck, Stephen R.

2010-01-01

Purpose. Dendritic cells (DCs) are antigen-presenting cells vital for initiating immune responses. In this study the authors examined the in vivo migratory capability of resident corneal DCs to various stimuli. Methods. The authors used mice expressing enhanced yellow fluorescent protein (eYFP) under control of the CD11c promoter to visualize corneal DCs. To assess the distribution and mobility of DCs, normal corneas were imaged in vivo and ex vivo with fluorescence microscopy. Intravital microscopy was used to examine the responses of resident central and peripheral corneal DCs to silver nitrate injury, lipopolysaccharide, microspheres, and tumor necrosis factor (TNF-α). In some experiments, TNF-α injection was used to first induce centripetal migration of DCs to the central cornea, which was subsequently reinjected with microspheres. Results. In normal corneas, DCs were sparsely distributed centrally and were denser in the periphery, with epithelial-level DCs extending into the epithelium. Videomicroscopy showed that though cell processes were in continuous movement, cells generally did not migrate. Within the first 6 hours after stimulation, neither central nor peripheral corneal DCs exhibited significant lateral migration, but central corneal DCs assumed extreme morphologic changes. An increased number of DCs in the TNF-α–stimulated central cornea were responsive to subsequent microsphere injection by adopting a migratory behavior, but not with increased speed. Conclusions. In vivo imaging reveals minimal lateral migration of corneal DCs after various stimuli. In contrast, DCs within the central cornea after initial TNF-α injection are more likely to respond to a secondary insult with lateral migration. PMID:20007837

Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Fallet, Clément; Caron, Julien; Oddos, Stephane; Tinevez, Jean-Yves; Moisan, Lionel; Sirat, Gabriel Y.; Braitbart, Philippe O.; Shorte, Spencer L.

2014-08-01

We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon taking place when a polarized beam is diffracted through a biaxial crystal. The illumination patterns generated by conical diffraction are more compact than the classical Gaussian beam; we use them to generate a super-resolution imaging modality. Conical Diffraction Microscopy (CODIM) resolution enhancement can be achieved with any type of objective on any kind of sample preparation and standard fluorophores. Conical diffraction can be used in multiple fashion to create new and disruptive technologies for super-resolution microscopy. This paper will focus on the first one that has been implemented and give a glimpse at what the future of microscopy using conical diffraction could be.

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies.

PubMed

Belsey, Natalie A; Garrett, Natalie L; Contreras-Rojas, L Rodrigo; Pickup-Gerlaugh, Adam J; Price, Gareth J; Moger, Julian; Guy, Richard H

2014-01-28

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Self-assembled nanotubes from single fluorescent amino acid

NASA Astrophysics Data System (ADS)

Babar, Dipak Gorakh; Sarkar, Sabyasachi

2017-04-01

Self-assembly of biomolecules has gained increasing attention as it generates various supramolecular structural assemblies having potential applications principally in biomedical sciences. Here, we show that amino acid like tryptophan or tyrosine readily aggregates as nanotubes via a simple self-assembly process. These were characterized by FTIR, scanning electron microscopy, and by fluorescence microscopy. Nanotubes prepared from tryptophan are having 200 nm inner diameter and those from tyrosine are having the same around 50 nm diameter.

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

PubMed Central

Shima, David T.; Haldar, Kasturi; Pepperkok, Rainer; Watson, Rose; Warren, Graham

1997-01-01

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus. PMID:9182657

Detection of Iss and Bor on the surface of Escherichia coli.

PubMed

Lynne, A M; Skyberg, J A; Logue, C M; Nolan, L K

2007-03-01

To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.

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.

Diving under a microscope--a new simple and versatile in vitro diving device for fluorescence and confocal microscopy allowing the controls of hydrostatic pressure, gas pressures, and kinetics of gas saturation.

PubMed

Wang, Qiong; Belhomme, Marc; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Theron, Michaël

2013-06-01

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

A portable microscopy system for fluorescence, polarized, and brightfield imaging

NASA Astrophysics Data System (ADS)

Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard

2018-02-01

The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

Mapping the distribution of emissive molecules in human ocular lipofuscin granules with near-field scanning optical microscopy.

PubMed

Krogmeier, J R; Clancy, C M; Pawlak, A; Rozanowska, M; Sarna, T; Simon, J D; Dunn, R C

2001-05-01

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age-related macular degeneration, recent attention has focused on the orange-emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near-field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.

The Relationship between Fenestrations, Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

PubMed Central

McNerney, Gregory P.; Owen, Dylan M.; Zencak, Dusan; Zykova, Svetlana N.; Crane, Harry; Huser, Thomas; Quinn, Ronald J.; Smedsrød, Bård; Le Couteur, David G.; Cogger, Victoria C.

2012-01-01

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished. PMID:23029409

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

PubMed

Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM)

PubMed Central

Beausang, John F.; Sun, Yujie; Quinlan, Margot E.; Forkey, Joseph N.; Goldman, Yale E.

2013-01-01

In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin. PMID:22550303

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

NASA Astrophysics Data System (ADS)

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-03-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers.

PubMed

Haring, Martijn T; Liv, Nalan; Zonnevylle, A Christiaan; Narvaez, Angela C; Voortman, Lenard M; Kruit, Pieter; Hoogenboom, Jacob P

2017-03-02

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.

PubMed

Model, Michael A; Blank, James L

2006-10-01

To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

PubMed Central

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-01-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample. PMID:28252673

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael, Jr.; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group.

PubMed

Kolmakov, Kirill; Wurm, Christian; Sednev, Maksim V; Bossi, Mariano L; Belov, Vladimir N; Hell, Stefan W

2012-03-01

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group.

Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy▿

PubMed Central

Zhang, Pengfei; Kong, Lingbo; Wang, Guiwen; Setlow, Peter; Li, Yong-qing

2011-01-01

Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+ with dipicolinic acid (CaDPA) was released rapidly at a highly variable time Tlag, the levels of spore nucleic acids remained nearly unchanged, and the Tlag times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before Tlag and reached maximum at a time slightly later than Trelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment. PMID:21602365

Fluorescent speckle microscopy of microtubules: how low can you go?

PubMed

Waterman-Storer, C M; Salmon, E D

1999-12-01

Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform "fluorescent speckle" pattern. Fluorescent speckle movements in time-lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- and 3-dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of approximately 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (approximately 0.27 microm) in the microscope. This small fraction of labeled subunits significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.

Fluorescence Lifetime Imaging Microscopy (FLIM) of quantum dots in living cells

NASA Astrophysics Data System (ADS)

Nadeau, Jay; Carlini, Lina

2013-02-01

Fluorescence lifetime imaging microscopy (FLIM) is an emerging imaging technique that can indicate environmental factors such as pH and redox potential by the effect of these factors on the fluorescence lifetimes of fluorophores. Semiconductor quantum dots (QDs) are highly sensitive to environment and so are ideal for use in FLIM, although certain experimental parameters must be carefully considered for QD imaging to account for their long lifetimes and two-photon behavior. We image the uptake of three types of QDs in cultured fibroblasts and show some preliminary results on the effects of endosomes and lysosomes on QD lifetimes. These results indicate the feasibility of FLIM for studies using QDs in live cells.

Combining single-molecule manipulation and single-molecule detection.

PubMed

Cordova, Juan Carlos; Das, Dibyendu Kumar; Manning, Harris W; Lang, Matthew J

2014-10-01

Single molecule force manipulation combined with fluorescence techniques offers much promise in revealing mechanistic details of biomolecular machinery. Here, we review force-fluorescence microscopy, which combines the best features of manipulation and detection techniques. Three of the mainstay manipulation methods (optical traps, magnetic traps and atomic force microscopy) are discussed with respect to milestones in combination developments, in addition to highlight recent contributions to the field. An overview of additional strategies is discussed, including fluorescence based force sensors for force measurement in vivo. Armed with recent exciting demonstrations of this technology, the field of combined single-molecule manipulation and single-molecule detection is poised to provide unprecedented views of molecular machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

PubMed Central

Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

2016-01-01

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

Synthesis and optical properties of water-soluble biperylene-based dendrimers.

PubMed

Shao, Pin; Jia, Ningyang; Zhang, Shaojuan; Bai, Mingfeng

2014-05-30

We report the synthesis and photophysical properties of three biperylene-based dendrimers, which show red fluorescence in water. A fluorescence microscopy study demonstrated uptake of biperylene-based dendrimers in living cells. Our results indicate that these biperylene-based dendrimers are promising candidates in fluorescence imaging applications with the potential as therapeutic carriers.

In vivo labeling of cortical astrocytes with sulforhodamine 101 (SR101).

PubMed

Nimmerjahn, Axel; Helmchen, Fritjof

2012-03-01

Fluorescent markers that stain particular cell types in the intact brain are essential tools for fluorescence microscopy because they enable studies of structure and function of cells identified in this way. Although cell type-specific fluorescence staining can be achieved through promoter-driven expression of fluorescent proteins, this genetic approach is generally labor- and cost-intensive. Alternative viral approaches for targeted fluorophore expression are relatively invasive. For astrocytes, there is a simple alternative. This protocol describes an easy and robust method for rapid (within minutes) and high-contrast staining of astrocytes in defined regions of the intact rodent cortex using the synthetic, water-soluble but non-fixable red fluorescent dye sulforhodamine 101 (SR101). Selective staining is achieved through local uptake and gap junction-mediated spread of SR101 following its topical application or injection into tissue. Applications, technical pitfalls, and limitations of the SR101-staining technique are discussed. Given its simplicity and reliability, SR101 staining is a valuable tool for the study of astrocyte function in the intact brain and for in vivo fluorescence microscopy in general.

Cellular uptake and trafficking of polydiacetylene micelles

NASA Astrophysics Data System (ADS)

Gravel, Edmond; Thézé, Benoit; Jacques, Isabelle; Anilkumar, Parambath; Gombert, Karine; Ducongé, Frédéric; Doris, Eric

2013-02-01

Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells.Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells. Electronic supplementary information (ESI) available: Detailed synthetic procedures and supplementary figures. See DOI: 10.1039/c2nr34149b

One-Step Synthesis of Fluorescent Boron Nitride Quantum Dots via a Hydrothermal Strategy Using Melamine as Nitrogen Source for the Detection of Ferric Ions.

PubMed

Huo, Bingbing; Liu, Bingping; Chen, Tao; Cui, Liang; Xu, Gengfang; Liu, Mengli; Liu, Jingquan

2017-10-10

A facile and effective approach for the preparation of functionalized born nitride quantum dots (BNQDs) with blue fluorescence was explored by the hydrothermal treatment of the mixture of boric acid and melamine at 200 °C for 15 h. The as-prepared BNQDs were characterized by transmission electron microscopy (TEM), high-resolution TEM, atomic force microscopy, X-ray photoelectron spectroscopy, UV-vis spectroscopy, and fluorescence spectroscopy. The single layered BNQDs with the average size of 3 nm showed a blue light emission under the illumination of the UV light. The BNQDs could be easily dispersed in an aqueous medium and applied as fluorescent probes for selective detection of Fe 3+ with remarkable selectivity and sensitivity (the lowest detection limit was 0.3 μM). The fluorescence fiber imaging demonstrated that the as-prepared quantum dots could be used as a valuable fluorchrome. Therefore, the BNQDs could be envisioned for potential applications in many fields such as biocompatible staining, fluorescent probes, and biological labeling.

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

PubMed

Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

2015-04-29

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

PubMed

Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

2018-04-26

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

PubMed

Chanoca, Alexandra; Burkel, Brian; Kovinich, Nik; Grotewold, Erich; Eliceiri, Kevin W; Otegui, Marisa S

2016-12-01

Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τ m ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τ m than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology

PubMed Central

Littlejohn, George R.; Mansfield, Jessica C.; Christmas, Jacqueline T.; Witterick, Eleanor; Fricker, Mark D.; Grant, Murray R.; Smirnoff, Nicholas; Everson, Richard M.; Moger, Julian; Love, John

2014-01-01

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the “negative space” within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells. PMID:24795734

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

Mechanism and applications of new fluorescent compounds produced by femtosecond laser surgery in biological tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Qu, Jianan Y.; Sun, Qiqi

2017-02-01

The single or multi-photon microscopy based on fluorescent labelling and staining is a sensitive and quantitative method that is widely used in molecular biology and medical research for a variety of experimental, analytical, and quality control applications. However, label-free method is highly desirable in biology and medicine when performing long term live imaging of biological system and obtaining instant tissue examination during surgery procedures. Recently, our group found that femtosecond laser surgery turned a variety of biological tissues and protein samples into highly fluorescent substances. The newly formed fluorescent compounds produced during the laser surgery can be excited via single- and two-photon processes over broad wavelength ranges. We developed a combined confocal and two-photon spectroscopic microscope to characterize the fluorescence from the new compound systematically. The structures of the femtosecond laser treated tissue were studied using Raman spectroscopy and transmission electron microscopy. Our study revealed the mechanisms of the fluorescence emission form the new compound. Furthermore, we demonstrated the applications of the fluorescent compounds for instant evaluation of femtosecond laser microsurgery, study of stem cell responses to muscle injury and neuro-regeneration after spinal cord injury.

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

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

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2015-02-24

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

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

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and similar to 90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context.« less

Noninvasive multi–photon fluorescence microscopy resolves retinol and retinal–condensation products in mouse eyes

PubMed Central

Palczewska, Grazyna; Maeda, Tadao; Imanishi, Yoshikazu; Sun, Wenyu; Chen, Yu; Williams, David R.; Piston, David; Maeda, Akiko; Palczewski, Krzysztof

2010-01-01

Multi–photon excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in sub–cellular structures called retinosomes mediate regeneration of the visual chromophore, 11–cis–retinal, by the visual cycle. But harmful fluorescent condensation products were also identified previously. We report that in wild type mice, excitation with λ ~730 nm identified retinosomes in the retinal pigment epithelium, whereas excitation with λ ~910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older WT mice and mice with defective clearance of all–trans–retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age–related macular degeneration and other defects in retinoid metabolism. PMID:21076393

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

PubMed Central

Peddie, Christopher J.; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O׳Toole, Peter; Larijani, Banafshe; Collinson, Lucy M.

2014-01-01

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. PMID:24637200

Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

PubMed

Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

2016-08-24

Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.

Enhanced emission of fluorophores on shrink-induced wrinkled composite structures.

PubMed

Sharma, Himanshu; Digman, Michelle A; Felsinger, Natasha; Gratton, Enrico; Khine, Michelle

2014-01-01

We introduce a manufacturable and scalable method for creating tunable wrinkled ferromagnetic-metallic structures to enhance fluorescence signals. Thin layers of nickel (Ni) and gold (Au) were deposited onto a pre-stressed thermoplastic (shrink wrap film) polymer. Heating briefly forced the metal films to buckle when the thermoplastic retracted, resulting in multi-scale composite 'wrinkles'. This is the first demonstration of leveraging the plasmons in such hybrid nanostructures by metal enhanced fluorescence (MEF) in the near-infrared wavelengths. We observed more than three orders of magnitude enhancement in the fluorescence signal of a single molecule of goat anti-mouse immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate, FITC, (FITC-IgG) by two-photon excitation with these structures. These large enhancements in the fluorescence signal at the nanoscale gaps between the composite wrinkles corresponded to shortened lifetimes due to localized surface plasmons. To characterize these structures, we combined fluctuation correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), and two-photon microscopy to spatially and temporally map the hot spots with high resolution.

Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

PubMed Central

Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

2016-01-01

Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

Polarization sensitive localization based super-resolution microscopy with a birefringent wedge

NASA Astrophysics Data System (ADS)

Sinkó, József; Gajdos, Tamás; Czvik, Elvira; Szabó, Gábor; Erdélyi, Miklós

2017-03-01

A practical method has been presented for polarization sensitive localization based super-resolution microscopy using a birefringent dual wedge. The measurement of the polarization degree at the single molecule level can reveal the chemical and physical properties of the local environment of the fluorescent dye molecule and can hence provide information about the sub-diffraction sized structure of biological samples. Polarization sensitive STORM imaging of the F-Actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.

Optical coherent tomography and fluorescent microscopy for the study of meningeal lymphatic systems

NASA Astrophysics Data System (ADS)

Semyachkina-Glushkovskaya, O.; Abdurashitov, A.; Namykin, A.; Fedosov, I.; Pavlov, A.; Karavaev, A.; Sindeeva, O.; Shirokov, A.; Ulanova, M.; Shushunova, N.; Khorovodov, A.; Agranovich, I.; Bodrova, A.; Sagatova, M.; Shareef, Ali Esmat; Saranceva, E.; Dvoryatkina, M.; Tuchin, V.

2018-04-01

The development of novel technologies for the imaging of meningeal lymphatic vessels is one of the amazing trends of biophotonics thanks to discovery of brain lymphatics over several years ago. However, there is the limited technologies exist for the study of lymphatics in vivo because lymphatic vessels are transparent with a low speed flow of lymph. Here we demonstrate the successful application of fluorescent microscopy for the imaging of lymphatic system in the mouse brain in vivo.

Motion estimation of subcellular structures from fluorescence microscopy images.

PubMed

Vallmitjana, A; Civera-Tregon, A; Hoenicka, J; Palau, F; Benitez, R

2017-07-01

We present an automatic image processing framework to study moving intracellular structures from live cell fluorescence microscopy. The system includes the identification of static and dynamic structures from time-lapse images using data clustering as well as the identification of the trajectory of moving objects with a probabilistic tracking algorithm. The method has been successfully applied to study mitochondrial movement in neurons. The approach provides excellent performance under different experimental conditions and is robust to common sources of noise including experimental, molecular and biological fluctuations.

Engineering cell-fluorescent ion track hybrid detectors

PubMed Central

2013-01-01

Background The lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al2O3:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). Methods The biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u−1). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information. Results The FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information. Conclusions The Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy resolution. PMID:23758749

Green synthesis of multifunctional carbon dots from coriander leaves and their potential application as antioxidants, sensors and bioimaging agents.

PubMed

Sachdev, Abhay; Gopinath, P

2015-06-21

In the present study, a facile one-step hydrothermal treatment of coriander leaves for preparing carbon dots (CDs) has been reported. Optical and structural properties of the CDs have been extensively studied by UV-visible and fluorescence spectroscopic, microscopic (transmission electron microscopy, scanning electron microscopy) and X-ray diffraction techniques. Surface functionality and composition of the CDs have been illustrated by elemental analysis and Fourier transform infrared spectroscopy (FTIR). Quenching of the fluorescence of the CDs in the presence of metal ions is of prime significance, hence CDs have been used as a fluorescence probe for sensitive and selective detection of Fe(3+) ions. Eventually, biocompatibility and bioimaging aspects of CDs have been evaluated on lung normal (L-132) and cancer (A549) cell lines. Qualitative analysis of cellular uptake of CDs has been pursued through fluorescence microscopy, while quantitative analysis using a flow cytometer provided an insight into the concentration and cell-type dependent uptake of CDs. The article further investigates the antioxidant activity of CDs. Therefore, we have validated the practicality of CDs obtained from a herbal carbon source for versatile applications.

In-vivo immunofluorescence confocal microscopy of herpes simplex virus type 1 keratitis

NASA Astrophysics Data System (ADS)

Kaufman, Stephen C.; Laird, Jeffery A.; Beuerman, Roger W.

1996-05-01

The white-light confocal microscope offers an in vivo, cellular-level resolution view of the cornea. This instrument has proven to be a valuable research and diagnostic tool for the study of infectious keratitis. In this study, we investigate the direct visualization of herpes simplex virus type 1 (HSV-1)-infected corneal epithelium, with in vivo confocal microscopy, using HSV-1 immunofluorescent antibodies. New Zealand white rabbits were infected with McKrae strain of HSV-1 in one eye; the other eye of each rabbit was used as an uninfected control. Four days later, the rabbits were anesthetized and a cellulose sponge was applied to each cornea, and a drop of direct HSV fluorescein-tagged antibody was placed on each sponge every 3 to 5 minutes for 1 hour. Fluorescence confocal microscopy was then performed. The HSV-infected corneas showed broad regions of hyperfluorescent epithelial cells. The uninfected corneas revealed no background fluorescence. Thus, using the confocal microscope with a fluorescent cube, we were able to visualize HSV-infected corneal epithelial cells tagged with a direct fluorescent antibody. This process may prove to be a useful clinical tool for the in vivo diagnosis of HSV keratitis.

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

Nanoscale cellular imaging with scanning angle interference microscopy.

PubMed

DuFort, Christopher; Paszek, Matthew

2014-01-01

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate. © 2014 Elsevier Inc. All rights reserved.

HDL particles incorporate into lipid bilayers - a combined AFM and single molecule fluorescence microscopy study.

PubMed

Plochberger, Birgit; Röhrl, Clemens; Preiner, Johannes; Rankl, Christian; Brameshuber, Mario; Madl, Josef; Bittman, Robert; Ros, Robert; Sezgin, Erdinc; Eggeling, Christian; Hinterdorfer, Peter; Stangl, Herbert; Schütz, Gerhard J

2017-11-21

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) - a main carrier of cholesterol in the blood stream - to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.

A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation.

PubMed

Pelet, S; Previte, M J R; Laiho, L H; So, P T C

2004-10-01

Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits. Copyright 2004 Biophysical Society

Local Intratracheal Delivery of Perfluorocarbon Nanoparticles to Lung Cancer Demonstrated with Magnetic Resonance Multimodal Imaging

PubMed Central

Wu, Lina; Wen, Xiaofei; Wang, Xiance; Wang, Chunan; Sun, Xilin; Wang, Kai; Zhang, Huiying; Williams, Todd; Stacy, Allen J.; Chen, Junjie; Schmieder, Anne H.; Lanza, Gregory M.; Shen, Baozhong

2018-01-01

Eighty percent of lung cancers originate as subtle premalignant changes in the airway mucosal epithelial layer of bronchi and alveoli, which evolve and penetrate deeper into the parenchyma. Liquid-ventilation, with perfluorocarbons (PFC) was first demonstrated in rodents in 1966 then subsequently applied as lipid-encapsulated PFC emulsions to improve pulmonary function in neonatal infants suffering with respiratory distress syndrome in 1996. Subsequently, PFC nanoparticles (NP) were extensively studied as intravenous (IV) vascular-constrained nanotechnologies for diagnostic imaging and targeted drug delivery applications. Methods: This proof-of-concept study compared intratumoral localization of fluorescent paramagnetic (M) PFC NP in the Vx2 rabbit model using proton (1H) and fluorine (19F) magnetic resonance (MR) imaging (3T) following intratracheal (IT) or IV administration. MRI results were corroborated by fluorescence microscopy. Results: Dynamic 1H-MR and 19F-MR images (3T) obtained over 72 h demonstrated marked and progressive accumulation of M-PFC NP within primary lung Vx2 tumors during the first 12 h post IT administration. Marked 1H and 19F MR signal persisted for over 72 h. In contradistinction, IV M-PFC NP produced a modest transient signal during the initial 2 h post-injection that was consistent circumferential blood pool tumor enhancement. Fluorescence microscopy of excised tumors corroborated the MR results and revealed enormous intratumor NP deposition on day 3 after IT but not IV treatment. Rhodamine-phospholipid incorporated into the PFC nanoparticle surfactant was distributed widely within the tumor on day 3, which is consistent with a hemifusion-based contact drug delivery mechanism previously reported. Fluorescence microscopy also revealed similar high concentrations of M-PFC NP given IT for metastatic Vx2 lung tumors. Biodistribution studies in mice revealed that M-PFC NP given IV distributed into the reticuloendothelial organs, whereas, the same dosage given IT was basically not detected beyond the lung itself. PFC NP given IT did not impact rabbit behavior or impair respiratory function. PFC NP effects on cells in culture were negligible and when given IV or IT no changes in rabbit hematology nor serum clinical chemistry parameters were measured. Conclusion: IT delivery of PFC NP offered unique opportunity to locally deliver PFC NP in high concentrations into lung cancers with minimal extratumor systemic exposure. PMID:29290827

Chapter 7: Total internal reflection fluorescence microscopy.

PubMed

Axelrod, Daniel

2008-01-01

Total internal reflection fluorescence microscopy (TIRFM), also known as evanescent wave microscopy, is used in a wide range of applications, particularly to view single molecules attached to planar surfaces and to study the position and dynamics of molecules and organelles in living culture cells near the contact regions with the glass coverslip. TIRFM selectively illuminates fluorophores only in a very thin (less than 100 nm deep) layer near the substrate, thereby avoiding excitation of fluorophores outside this subresolution optical section. This chapter reviews the history, current applications in cell biology and biochemistry, basic optical theory, combinations with numerous other optical and spectroscopic approaches, and a range of setup methods, both commercial and custom.

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Nanometer-Scale Dissection of Chromosomes by Atomic Force Microscopy Combined with Heat-Denaturing Treatment

NASA Astrophysics Data System (ADS)

Tsukamoto, Kazumi; Kuwazaki, Seigo; Yamamoto, Kimiko; Shichiri, Motoharu; Yoshino, Tomoyuki; Ohtani, Toshio; Sugiyama, Shigeru

2006-03-01

We have developed a method for dissecting chromosome fragments with a size of a few hundred nanometers by atomic force microscopy (AFM). By using this method, we demonstrated reproducible dissections of silkworm chromosomes in the pachytene phase. The dissected fragments were successfully recovered on the cantilever tips, as confirmed by fluorescent microscopy using fluorescent stained chromosomes. To recover dissected chromosome fragments from a larger chromosome, such as the human metaphase chromosome of a somatic cell, heat denaturation was found to be effective. Further improvements in this method may lead to a novel tool for isolating valuable genes and/or investigating local genome structures in the near future.

Improving nanoparticle dispersion and charge transfer in cadmium telluride tetrapod and conjugated polymer blends.

PubMed

Monson, Todd C; Hollars, Christopher W; Orme, Christine A; Huser, Thomas

2011-04-01

The dispersion of CdTe tetrapods in a conducting polymer and the resulting charge transfer is studied using a combination of confocal fluorescence microscopy and atomic force microscopy (AFM). The results of this work show that both the tetrapod dispersion and charge transfer between the CdTe and conducting polymer (P3HT) are greatly enhanced by exchanging the ligands on the surface of the CdTe and by choosing proper solvent mixtures. The ability to experimentally probe the relationship between particle dispersion and charge transfer through the combination of AFM and fluorescence microscopy provides another avenue to assess the performance of polymer/semiconductor nanoparticle composites. © 2011 American Chemical Society

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

Imaging of surgical margin in pancreatic metastasis using two-photon excited fluorescence microscopy

NASA Astrophysics Data System (ADS)

Chen, Jing; Hong, Zhipeng; Chen, Hong; Chen, Youting; Xu, Yahao; Zhu, Xiaoqin; Zhuo, Shuangmu; Shi, Zheng; Chen, Jianxin

2014-09-01

Two-photon excited fluorescence (TPEF) microscopy, has become a powerful tool for imaging unstained tissue samples at subcellular level in biomedical research. The purpose of this study was to determine whether TPEF imaging of histological sections without H-E staining can be used to identify the boundary between normal pancreas and pancreatic metastasis from renal cell carcinoma (RCC). The typical features such as the significant increase of cancerous nests, the absence of pancreatic ductal, the appearance of cancer cells were observed to present the boundary between normal pancreas and pancreatic metastasis from RCC. These results correlated well with the corresponding histological outcomes. With the advent of clinically miniaturized TPEF microscopy and integrative endoscopy, TPEF microscopy has the potential application on surgical location of pancreatic metastasis from RCC in the near future.

Non-rigid multi-frame registration of cell nuclei in live cell fluorescence microscopy image data.

PubMed

Tektonidis, Marco; Kim, Il-Han; Chen, Yi-Chun M; Eils, Roland; Spector, David L; Rohr, Karl

2015-01-01

The analysis of the motion of subcellular particles in live cell microscopy images is essential for understanding biological processes within cells. For accurate quantification of the particle motion, compensation of the motion and deformation of the cell nucleus is required. We introduce a non-rigid multi-frame registration approach for live cell fluorescence microscopy image data. Compared to existing approaches using pairwise registration, our approach exploits information from multiple consecutive images simultaneously to improve the registration accuracy. We present three intensity-based variants of the multi-frame registration approach and we investigate two different temporal weighting schemes. The approach has been successfully applied to synthetic and live cell microscopy image sequences, and an experimental comparison with non-rigid pairwise registration has been carried out. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Crystallization Kinetics of an Amorphous Pharmaceutical Compound Using Fluorescence-Lifetime-Imaging Microscopy.

PubMed

Rautaniemi, Kaisa; Vuorimaa-Laukkanen, Elina; Strachan, Clare J; Laaksonen, Timo

2018-05-07

Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence-lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and γ- and α-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 °C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.

Frequency division multiplexed multi-color fluorescence microscope system

NASA Astrophysics Data System (ADS)

Le, Vu Nam; Yang, Huai Dong; Zhang, Si Chun; Zhang, Xin Rong; Jin, Guo Fan

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame rate is consistent with the frame rate of the camera. The optical system is simpler and does not need extra color separation element. In addition, this method has a good filtering effect on the ambient light or other light signals which are not affected by the modulation process.