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.

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.

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.

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

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.

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.

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

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.

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

PubMed

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

2006-01-01

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

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.

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

Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

PubMed

Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

2014-03-01

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

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

PubMed Central

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

2016-01-01

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

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.

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

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.

Near-infrared branding efficiently correlates light and electron microscopy.

PubMed

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

2011-06-05

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

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.

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.

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

PubMed

Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

2015-06-11

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

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.

Surface molecular imprinting onto fluorescein-coated magnetic nanoparticlesvia reversible addition fragmentation chain transfer polymerization: A facile three-in-one system for recognition and separation of endocrine disrupting chemicals

NASA Astrophysics Data System (ADS)

Li, Ying; Dong, Cunku; Chu, Jia; Qi, Jingyao; Li, Xin

2011-01-01

In this study, we present a general protocol for the making of surface-imprinted magnetic fluorescence beads viareversible addition-fragmentation chain transfer polymerization. The resulting composites were characterized by X-ray diffraction analysis, transmission electron microscopy, scanning electron microscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy. The as-synthesized beads exhibited homogeneous polymer films (thickness of about 5.7 nm), spherical shape, high fluorescence intensity and magnetic property (Magnetization (Ms) = 3.67 emu g-1). The hybrids bind the original template 17β-estradiol with an appreciable selectivity over structurally related compounds. In addition, the resulting hybrids performed without obvious deterioration after five repeated cycles. This study therefore demonstrates the potential of molecularly imprinted polymers for the recognition and separation of endocrine disrupting chemicals.In this study, we present a general protocol for the making of surface-imprinted magnetic fluorescence beads viareversible addition-fragmentation chain transfer polymerization. The resulting composites were characterized by X-ray diffraction analysis, transmission electron microscopy, scanning electron microscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy. The as-synthesized beads exhibited homogeneous polymer films (thickness of about 5.7 nm), spherical shape, high fluorescence intensity and magnetic property (Magnetization (Ms) = 3.67 emu g-1). The hybrids bind the original template 17β-estradiol with an appreciable selectivity over structurally related compounds. In addition, the resulting hybrids performed without obvious deterioration after five repeated cycles. This study therefore demonstrates the potential of molecularly imprinted polymers for the recognition and separation of endocrine disrupting chemicals. Electronic supplementary information (ESI) available: Supplementary figure S1. The hysteresis loop of Fe3O4 (a), Fe3O4@SiO2 (b), and Fe3O4@SiO2-Dye-SiO2 (c). See DOI: 10.1039/c0nr00614a

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.

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.

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.

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

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.

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).

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.

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-03-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. (c) 2010 Elsevier Inc. All rights reserved.

Preparation, characterization, physical properties, and photoconducting behaviour of anthracene derivative nanowires

NASA Astrophysics Data System (ADS)

Xiao, Jinchong; Yin, Zongyou; Yang, Bo; Liu, Yi; Ji, Li; Guo, Jun; Huang, Ling; Liu, Xuewei; Yan, Qingyu; Zhang, Hua; Zhang, Qichun

2011-11-01

Organic nanowires of 9,10-dibromoanthracene (DBA) and 9,10-dicyanoanthracene (DCNA) were obtained by adding the THF solution of DBA/DCNA into water containing P123 surfactants. The as-prepared nanowires were characterized by UV-vis, fluorescence spectra, Field Emission Scanning Electron Microscopy (FESEM), and Transmission Electron Microscopy (TEM). We found that DBA and DCNA nanowires emitted green light rather than blue light for molecules in THF solution. The red-shift UV and fluorescent spectra of DBA and DCNA nanowires implied that these nanowires were formed through J-aggregation. The photoconducting study of DBA/DCNA nanowire-based network on rGO/SiO2/Si shows different photocurrent behaviors upon irradiation, which displayed that electron transfer from DCNA nanowire to rGO was stronger than that of DBA nanowires to rGO.Organic nanowires of 9,10-dibromoanthracene (DBA) and 9,10-dicyanoanthracene (DCNA) were obtained by adding the THF solution of DBA/DCNA into water containing P123 surfactants. The as-prepared nanowires were characterized by UV-vis, fluorescence spectra, Field Emission Scanning Electron Microscopy (FESEM), and Transmission Electron Microscopy (TEM). We found that DBA and DCNA nanowires emitted green light rather than blue light for molecules in THF solution. The red-shift UV and fluorescent spectra of DBA and DCNA nanowires implied that these nanowires were formed through J-aggregation. The photoconducting study of DBA/DCNA nanowire-based network on rGO/SiO2/Si shows different photocurrent behaviors upon irradiation, which displayed that electron transfer from DCNA nanowire to rGO was stronger than that of DBA nanowires to rGO. Electronic supplementary information (ESI) available: XRD patterns and simulations, and FT-IR spectra. CCDC reference numbers 840471. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c1nr10655d

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.

Symposium LL: Nanowires--Synthesis Properties Assembly and Application

DTIC Science & Technology

2010-09-10

dedicated hard x - ray microscopy beamline is operated in partnership with the Advanced Photon Source to provide fluorescence, diffraction, and...characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X - ray diffraction (XRD) measurements, proving it to be...Investigation of Preferred Growth Direction of GaN Nanorods by Synchrotron X - ray Reciprocal Space Mapping. Yuri Sohn1, Sanghwa Lee1, Chinkyo Kim1 and Dong

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.

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.

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.

Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-11-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae

PubMed Central

Misonou, Yoshiko; Kikuchi, Maiko; Sato, Hiroshi; Inai, Tomomi; Kuroiwa, Tsuneyoshi; Tanaka, Kenji; Miyakawa, Isamu

2014-01-01

ABSTRACT When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast. PMID:24771619

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

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

Characterization of the host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy, and immunological techniques

USGS Publications Warehouse

Bartholomew, J.L.; Smith, C.E.; Rohovec, J.S.; Fryer, J.L.

1989-01-01

The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta-susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

PubMed Central

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-01-01

Abstract. 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. PMID:26440760

Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

NASA Astrophysics Data System (ADS)

Seacor, Taylor; Howell, Carina

2013-03-01

Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

Cytocompatibility and uptake of halloysite clay nanotubes.

PubMed

Vergaro, Viviana; Abdullayev, Elshad; Lvov, Yuri M; Zeitoun, Andre; Cingolani, Roberto; Rinaldi, Ross; Leporatti, Stefano

2010-03-08

Halloysite is aluminosilicate clay with hollow tubular structure of 50 nm external diameter and 15 nm diameter lumen. Halloysite biocompatibility study is important for its potential applications in polymer composites, bone implants, controlled drug delivery, and for protective coating (e.g., anticorrosion or antimolding). Halloysite nanotubes were added to different cell cultures for toxicity tests. Its fluorescence functionalization by aminopropyltriethosilane (APTES) and with fluorescently labeled polyelectrolyte layers allowed following halloysite uptake by the cells with confocal laser scanning microscopy (CLSM). Quantitative Trypan blue and MTT measurements performed with two neoplastic cell lines model systems as a function of the nanotubes concentration and incubation time indicate that halloysite exhibits a high level of biocompatibility and very low cytotoxicity, rendering it a good candidate for household materials and medicine. A combination of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning force microscopy (SFM) imaging techniques have been employed to elucidate the structure of halloysite nanotubes.

Use of fluorescence and scanning electron microscopy as tools in teaching biology

NASA Astrophysics Data System (ADS)

Ghosh, Nabarun; Silva, Jessica; Vazquez, Aracely; Das, A. B.; Smith, Don W.

2011-06-01

Recent nationwide surveys reveal significant decline in students' interest in Math and Sciences. The objective of this project was to inspire young minds in using various techniques involved in Sciences including Scanning Electron Microscopy. We used Scanning Electron Microscope in demonstrating various types of Biological samples. An SEM Tabletop model in the past decade has revolutionized the use of Scanning Electron Microscopes. Using SEM Tabletop model TM 1000 we studied biological specimens of fungal spores, pollen grains, diatoms, plant fibers, dust mites, insect parts and leaf surfaces. We also used fluorescence microscopy to view, to record and analyze various specimens with an Olympus BX40 microscope equipped with FITC and TRITC fluorescent filters, a mercury lamp source, DP-70 digital camera with Image Pro 6.0 software. Micrographs were captured using bright field microscopy, the fluoresceinisothiocyanate (FITC) filter, and the tetramethylrhodamine (TRITC) filter settings at 40X. A high pressure mercury lamp or UV source was used to excite the storage molecules or proteins which exhibited autofluorescence. We used fluorescent microscopy to confirm the localization of sugar beet viruses in plant organs by viewing the vascular bundles in the thin sections of the leaves and other tissues. We worked with the REU summer students on sample preparation and observation on various samples utilizing the SEM. Critical Point Drying (CPD) and metal coating with the sputter coater was followed before observing some cultured specimen and the samples that were soft in textures with high water content. SEM Top allowed investigating the detailed morphological features that can be used for classroom teaching. Undergraduate and graduate researchers studied biological samples of Arthropods, pollen grains and teeth collected from four species of snakes using SEM. This project inspired the research students to pursue their career in higher studies in science and 45% of the undergraduates participated in this project entered Graduate school.

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

PubMed Central

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

2014-01-01

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

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.

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.

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

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.

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

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.

Efficient fluorescence quenching in electrochemically exfoliated graphene decorated with gold nanoparticles

NASA Astrophysics Data System (ADS)

Hurtado-Morales, M.; Ortiz, M.; Acuña, C.; Nerl, H. C.; Nicolosi, V.; Hernández, Y.

2016-07-01

High surface area graphene sheets were obtained by electrochemical exfoliation of graphite in an acid medium under constant potential conditions. Filtration and centrifugation processes played an important role in order to obtain stable dispersions in water. Scanning electron microscopy and transmission electron microscopy imaging revealed highly exfoliated crystalline samples of ∼5 μm. Raman, Fourier transform infrared and x-ray photoelectron spectroscopy further confirmed the high quality of the exfoliated material. The electrochemically exfoliated graphene (EEG) was decorated with gold nanoparticles (AuNPs) using sodium cholate as a buffer layer. This approach allowed for a non-covalent functionalization without altering the desirable electronic properties of the EEG. The AuNP-EEG samples were characterized with various techniques including absorbance and fluorescence spectroscopy. These samples displayed a fluorescence signal using an excitation wavelength of 290 nm. The calculated quantum yield (Φ) for these samples was 40.04%, a high efficiency compared to previous studies using solution processable graphene.

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

PubMed Central

Johnson, M. Brittany; Criss, Alison K.

2013-01-01

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

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.

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

PubMed Central

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

2009-01-01

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

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.

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.

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

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.

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

Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy.

PubMed

Kreplak, Laurent; Richter, Karsten; Aebi, Ueli; Herrmann, Harald

2008-01-01

Intermediate filaments (IFs) were originally discovered and defined by electron microscopy in myoblasts. In the following it was demonstrated and confirmed that they constitute, in addition to microtubules and microfilaments, a third independent, general filament system in the cytoplasm of most metazoan cells. In contrast to the other two systems, IFs are present in cells in two principally distinct cytoskeletal forms: (i) extended and free-running filament arrays in the cytoplasm that are integrated into the cytoskeleton by associated proteins of the plakin type; and (ii) a membrane- and chromatin-bound thin 'lamina' of a more or less regular network of interconnected filaments made from nuclear IF proteins, the lamins, which differ in several important structural aspects from cytoplasmic IF proteins. In man, more than 65 genes code for distinct IF proteins that are expressed during embryogenesis in various routes of differentiation in a tightly controlled manner. IF proteins exhibit rather limited sequence identity implying that the different types of IFs have distinct biochemical properties. Hence, to characterize the structural properties of the various IFs, in vitro assembly regimes have been developed in combination with different visualization methods such as transmission electron microscopy of fixed and negatively stained samples as well as methods that do not use staining such as scanning transmission electron microscopy (STEM) and cryoelectron microscopy as well as atomic force microscopy. Moreover, with the generation of both IF-type specific antibodies and chimeras of fluorescent proteins and IF proteins, it has become possible to investigate the subcellular organization of IFs by correlative fluorescence and electron microscopic methods. The combination of these powerful methods should help to further develop our understanding of nuclear architecture, in particular how nuclear subcompartments are organized and in which way lamins are involved.

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

Highly stable lipid-encapsulation of fluorescent nanodiamonds for bioimaging applications.

PubMed

Sotoma, Shingo; Hsieh, Feng-Jen; Chen, Yen-Wei; Tsai, Pei-Chang; Chang, Huan-Cheng

2018-01-23

Highly stable lipid-encapsulated fluorescent nanodiamonds (FNDs) are produced by photo-crosslinking of diacetylene-containing lipids physically attached to the FND surface. Not only is this coating method simple and fast, but also it gives the FND-lipid hybrids favorable properties for bioapplications. The hybrids are useful as fluorescent biolabels as well as fiducial markers for correlative light and electron microscopy.

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

PubMed Central

Barrasa, J. M.; Gutiérrez, A.; Escaso, V.; Guillén, F.; Martínez, M. J.; Martínez, A. T.

1998-01-01

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H2O2-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw. PMID:9435085

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.

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

Microcapsules on Streptococcus mutans serotypes by electron microscopy.

PubMed

Grenier, E M; Gray, R H; Loesche, W J; Eveland, W C

1977-02-01

Extracellular microcapsules have been demonstrated on cells of most serotypes of Streptococcus mutans by electron microscopy, using bacterial strains of the various serotypes and peroxidase labeled or unlabeled immune serum. A correlation was noted between the amount of capsular substance on the strains of S mutans examined and degree of antigenicity as expressed by the indirect fluorescent antibody (FA) title. A serotype d strain was shown to lose both antigenicity as determined by the FA reaction and capsular material as seen by electron microscopy with repeated in vitro passage. When 10% unheated rabbit serum was added to the medium, antigenicity and capsular material were restored.

Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

NASA Astrophysics Data System (ADS)

Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

2005-04-01

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

NASA Astrophysics Data System (ADS)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tong, Yongpeng; Li, Changming; Liang, Feng; Chen, Jianmin; Zhang, Hong; Liu, Guoqing; Sun, Huibin; Luong, John H. T.

2008-12-01

Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al 2O 3 and TiO 2) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl 2) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al 2O 3 and TiO 2 nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe 2O 3 nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

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.

Towards the Development of a Low-Cost Device for the Detection of Explosives Vapors by Fluorescence Quenching of Conjugated Polymers in Solid Matrices.

PubMed

Martelo, Liliana M; das Neves, Tiago F Pimentel; Figueiredo, João; Marques, Lino; Fedorov, Alexander; Charas, Ana; Berberan-Santos, Mário N; Burrows, Hugh D

2017-11-03

Conjugated polymers (CPs) have proved to be promising chemosensory materials for detecting nitroaromatic explosives vapors, as they quickly convert a chemical interaction into an easily-measured high-sensitivity optical output. The nitroaromatic analytes are strongly electron-deficient, whereas the conjugated polymer sensing materials are electron-rich. As a result, the photoexcitation of the CP is followed by electron transfer to the nitroaromatic analyte, resulting in a quenching of the light-emission from the conjugated polymer. The best CP in our studies was found to be poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-bithiophene] (F8T2). It is photostable, has a good absorption between 400 and 450 nm, and a strong and structured fluorescence around 550 nm. Our studies indicate up to 96% quenching of light-emission, accompanied by a marked decrease in the fluorescence lifetime, upon exposure of the films of F8T2 in ethyl cellulose to nitrobenzene (NB) and 1,3-dinitrobenzene (DNB) vapors at room temperature. The effects of the polymeric matrix, plasticizer, and temperature have been studied, and the morphology of films determined by scanning electron microscopy (SEM) and confocal fluorescence microscopy. We have used ink jet printing to produce sensor films containing both sensor element and a fluorescence reference. In addition, a high dynamic range, intensity-based fluorometer, using a laser diode and a filtered photodiode was developed for use with this system.

Fluorescence histochemical and elec-ron microscopical observations on sympathetic ganglia of the chick embryo cultured with and without hydrocortisone.

PubMed

Hervonen, H; Eränkö, O

1975-01-01

Lumbar sympathetic ganglia of 12-day-old chick embryos were cultured in organ cultures for 14 days with 1, 10 or 100 mg/l of hydrocortisone or without it. Catecholamines were demonstrated by the formaldehyde-induced fluorescence method. For electron microscopy, the cultures were fixed with glutarialdehyde and osmium tetroxide. Two types of cells with catecholamine fluoresecence were observed in the control cultures: (1) weakly fluorescent sympathetic neurons and sympathicoblasts with long nerve fibres, which were the most common cell type in the explant, and (2) brightly fluorescent cells with or without fluorescent processes, which were less common and were scattered in the explant. Hydrocortisone caused a great increase in the number of the brightly fluorescent cells. With 10 mg/l of hydrocortisone the increase was about ten-fold as compared with the control cultures. There was no change in the morphology of the cells, nor could any change be observed in the fluorescence intensity by eye. Electron microscopically the mature neurons were the most common cell type on the surface of the culture, while more immature sympathicoblasts were seen in the deeper layers. Cells were also found which contained large numbers of catecholamine-strong granular vesicles 105-275 nm in diameter. These cells were infrequent. They had round vesicular nuclei and resembled also in other respects sympathicoblasts or young nerve cells. One such cell was found in mitotic division by electron microscopy. Hydrocortisone caused a marked increase in the number of these granule-containing cells and their processes. Cells which could have been classified as the small intensely fluorescent cells of the mammalian ganglion type or their electron microscopic equivalent, the granule-containing cells were found neither in the control cultures nor in the hydrocortisone-containing cultures. It is concluded that most brightly fluorescent cells in cultured sympathetic ganglia of the chick are nerve cells or sympathicoblasts rich in amine-storing granular vesicles.

Effects of a common worldwide drink (Beer) on L-Phenylalanine and L-Tyrosine fibrillar assemblies

NASA Astrophysics Data System (ADS)

Banik, Debasis; Banerjee, Pavel; Sabeehuddin, Ghazi; Sarkar, Nilmoni

2017-11-01

In this letter, small amount of beer [0.42-2.08% (v/v)] is employed to investigate the fibril inhibition kinetics of 1 mM L-Phenylalanine and L-Tyrosine (relevant to disease condition) using Fluorescence Lifetime imaging Microscopy (FLIM), Field Emission Scanning Electron Microscopy (FESEM) and High Resolution Transmission Electron Microscopic (HR-TEM) techniques. Our results indicate that 1.67 and 0.42% of beer is sufficient for effective breakdown of L-Phe and L-Tyr assemblies, respectively. Quantitative information about fibril inhibition is obtained from Fluorescence Correlation Spectroscopic (FCS) measurements. We have shown that the morphology of L-Phe changes to L-Tyr in presence of 2,2‧-Bipyridine-3,3‧-diol (BP(OH)2).

Microscopy image segmentation tool: Robust image data analysis

NASA Astrophysics Data System (ADS)

Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

2014-03-01

We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABAA receptor clustering induced by inhibitory synaptic plasticity.

PubMed

Orlando, Marta; Ravasenga, Tiziana; Petrini, Enrica Maria; Falqui, Andrea; Marotta, Roberto; Barberis, Andrea

2017-10-23

Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABA A Receptors (GABA A Rs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABA A R clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABA A R clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.

As-prepared MoS2 quantum dot as a facile fluorescent probe for long-term tracing of live cells

NASA Astrophysics Data System (ADS)

Zhou, Kai; Zhang, Yue; Xia, Zhining; Wei, Weili

2016-07-01

Recently, the newly emerged two-dimensional nanomaterials, layered transition metal dichalcogenide (e.g. MoS2) nanosheets, have drawn tremendous attentions due to their extraordinary electronic and optical properties, and MoS2 quantum dots (MoS2 QDs) with lateral sizes less than 10 nm have been found to be highly luminescent. In the present study, a facile approach for large-scale preparation of MoS2 QDs by Na intercalation reaction without using any toxic organic reagents is proposed. MoS2 QDs were carefully characterized by various techniques including transmission electron microscopy, atomic force microscopy, dynamic light scattering, spectroscopy, in vitro cytotoxicology, and capillary electrophoresis. The as-prepared MoS2 QDs were strongly fluorescent, highly photo-stable, low in cytotoxicity, and readily reactive to thiols. These inherent properties of MoS2 QDs make them excellent fluorescent probes for long-term live cell tracing. The results of live cells imaging indicated that MoS2 QD stained cells remained highly fluorescent after long-term culture, and could be easily traced from other co-cultured cell lines.

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

PubMed Central

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

2012-01-01

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

Synthesis and characterization of arsenic-doped cysteine-capped thoria-based nanoparticles

NASA Astrophysics Data System (ADS)

Pereira, F. J.; Díez, M. T.; Aller, A. J.

2013-09-01

Thoria materials have been largely used in the nuclear industry. Nonetheless, fluorescent thoria-based nanoparticles provide additional properties to be applied in other fields. Thoria-based nanoparticles, with and without arsenic and cysteine, were prepared in 1,2-ethanediol aqueous solutions by a simple precipitation procedure. The synthesized thoria-based nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (ED-XRS), Raman spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and fluorescence microscopy. The presence of arsenic and cysteine, as well as the use of a thermal treatment facilitated fluorescence emission of the thoria-based nanoparticles. Arsenic-doped and cysteine-capped thoria-based nanoparticles prepared in 2.5 M 1,2-ethanediol solutions and treated at 348 K showed small crystallite sizes and strong fluorescence. However, thoria nanoparticles subjected to a thermal treatment at 873 K also produced strong fluorescence with a very narrow size distribution and much smaller crystallite sizes, 5 nm being the average size as shown by XRD and TEM. The XRD data indicated that, even after doping of arsenic in the crystal lattice of ThO2, the samples treated at 873 K were phase pure with the fluorite cubic structure. The Raman and FT-IR spectra shown the most characteristics vibrational peaks of cysteine together with other peaks related to the bonds of this molecule to thoria and arsenic when present.

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

PubMed

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

2014-01-01

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

5-Arylvinyl-2,2′-bipyridyls: Bright “push–pull” dyes as components in fluorescent indicators for zinc ions

PubMed Central

Zhu, Lei; Younes, Ali H.; Yuan, Zhao; Clark, Ronald J.

2015-01-01

This article reviews the zinc(II)-dependent photophysical properties of arylvinylbipyridines (AVBs), a class of fluoroionophores in which 2,2′-bipyridyl and an aryl moiety are electronically conjugated. Zinc(II) binding of an AVB may lead to an emission bathochromic shift of the fluoroionophore without diminishing its fluorescence quantum yield. This observation can be explained using the excited state model of electron donor–π bridge–electron acceptor “push–pull” fluorophores, in which the bipy moiety acts as an electron acceptor, and zinc(II)-coordination strengthens its electron affinity. The spectral sensitivity of bipy-containing fluoroionophores, such as AVBs, to zinc(II) can be exploited to prepare fluorescent indicators for this ion. In several cases, AVB moieties are incorporated in fluorescent heteroditopic ligands, so that the variation of zinc(II) concentration over a relatively large range can be correlated to fluorescence changes in either intensity or color. AVB fluoroionophores are also used to introduce an intramolecular Förster resonance energy transfer (FRET) strategy for creating zinc(II) indicators with high photostability and a narrow emission band, two desired characteristics of dyes used in fluorescence microscopy. PMID:26190906

Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

PubMed

Tücking, Katrin-Stephanie; Grützner, Verena; Unger, Ronald E; Schönherr, Holger

2015-07-01

The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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

PubMed

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

2015-05-07

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

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.

Fluorescence labeling of colloidal core-shell particles with defined isoelectric points for in vitro studies.

PubMed

Daberkow, Timo; Meder, Fabian; Treccani, Laura; Schowalter, Marco; Rosenauer, Andreas; Rezwan, Kurosch

2012-02-01

In the light of in vitro nanotoxicological studies fluorescence labeling has become standard for particle localization within the cell environment. However, fluorescent labeling is also known to significantly alter the particle surface chemistry and therefore potentially affect the outcome of cell studies. Hence, fluorescent labeling is ideally carried out without changing, for example, the isoelectric point. A simple and straightforward method for obtaining fluorescently labeled spherical metal oxide particles with well-defined isoelectric points and a narrow size distribution is presented in this study. Spherical amorphous silica (SiO2, 161 nm diameter) particles were used as the substrate material and were coated with silica, alumina (Al2O3), titania (TiO2), or zirconia (ZrO2) using sol-gel chemistry. Fluorescent labeling was achieved by directly embedding rhodamine 6G dye in the coating matrix without affecting the isoelectric point of the metal oxide coatings. The coating quality was confirmed by high resolution transmission electron microscopy, energy filtered transmission electron microscopy and electrochemical characterization. The coatings were proven to be stable for at least 240 h under different pH conditions. The well-defined fluorescent particles can be directly used for biomedical investigations, e.g. elucidation of particle-cell interactions in vitro. Copyright © 2011 Acta Materialia Inc. Published by 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

Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

PubMed Central

Zurbuchen, Mark A.; Lake, Michael P.; Kohan, Sirus A.; Leung, Belinda; Bouchard, Louis-S.

2013-01-01

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable “zooming-in” to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery. PMID:24036840

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.

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.

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

PubMed

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

2015-12-01

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

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Dual-labeling method for electron microscopy to characterize synaptic connectivity using genetically encoded fluorescent reporters in Drosophila

PubMed Central

Tanaka, Nobuaki K.; Dye, Louis; Stopfer, Mark

2010-01-01

Light and electron microscopy (LM and EM) both offer important advantages for characterizing neuronal circuitry in intact brains: LM can reveal the general patterns neurons trace between brain areas, and EM can confirm synaptic connections between identified neurons within a small area. In a few species, genetic labeling with fluorescent proteins has been used with LM to visualize many kinds of neurons and to analyze their morphologies and projection patterns. However, combining these large-scale patterns with the fine detail available in EM analysis has been a technical challenge. To analyze the synaptic connectivity of neurons expressing fluorescent markers with EM, we developed a dual-labeling method for use with pre-embedded brains. In Drosophila expressing genetic labels and also injected with markers we visualized synaptic connections among two populations of neurons in the AL, one of which has been shown to mediate a specific function, odor evoked neural oscillation. PMID:21074556

Some radiation effects on organic binders in X-ray fluorescence spectrometry

NASA Astrophysics Data System (ADS)

Novosel-Radović, Vj.; MaljkoviĆ, Da.; NenadiĆ, N.

The paper deals with diminished wear resistance of standard samples in X-ray fluorescence spectrometry. The effect of X-ray irradiation on pellet samples, pressed with starch as organic binder, was investigated by sieve analysis and scanning electron microscopy. A change in the starch grain size was found as a result of swelling and cracking.

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

PubMed

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

2017-01-01

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

Fluorescence of silicon nanoparticles prepared by nanosecond pulsed laser

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

Liu, Chunyang, E-mail: chunyangliu@126.com; Sui, Xin; Yang, Fang

2014-03-15

A pulsed laser fabrication method is used to prepare fluorescent microstructures on silicon substrates in this paper. A 355 nm nanosecond pulsed laser micromachining system was designed, and the performance was verified and optimized. Fluorescence microscopy was used to analyze the photoluminescence of the microstructures which were formed using the pulsed laser processing technique. Photoluminescence spectra of the microstructure reveal a peak emission around 500 nm, from 370 nm laser irradiation. The light intensity also shows an exponential decay with irradiation time, which is similar to attenuation processes seen in porous silicon. The surface morphology and chemical composition of themore » microstructure in the fabricated region was also analyzed with multifunction scanning electron microscopy. Spherical particles are produced with diameters around 100 nm. The structure is compared with porous silicon. It is likely that these nanoparticles act as luminescence recombination centers on the silicon surface. The small diameter of the particles modifies the band gap of silicon by quantum confinement effects. Electron-hole pairs recombine and the fluorescence emission shifts into the visible range. The chemical elements of the processed region are also changed during the interaction between laser and silicon. Oxidation and carbonization play an important role in the enhancement of fluorescence emission.« less

Three-dimensional Organization of pKi-67: A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold

PubMed Central

Cheutin, Thierry; O'Donohue, Marie-Françoise; Beorchia, Adrien; Klein, Christophe; Kaplan, Hervé; Ploton, Dominique

2003-01-01

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250–300 nm in diameter, which are themselves composed of 30–50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin. PMID:14566014

Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using FluoroNanogold.

PubMed

Cheutin, Thierry; O'Donohue, Marie-Françoise; Beorchia, Adrien; Klein, Christophe; Kaplan, Hervé; Ploton, Dominique

2003-11-01

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.

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 double responsive smart upconversion fluorescence sensing material for glycoprotein.

PubMed

Guo, Ting; Deng, Qiliang; Fang, Guozhen; Yun, Yaguang; Hu, Yongjin; Wang, Shuo

2016-11-15

A novel strategy was developed to prepare double responsive smart upconversion fluorescence material for highly specific enrichment and sensing of glycoprotein. The novel double responsive smart sensing material was synthesized by choosing Horse radish peroxidase (HRP) as modal protein, the grapheme oxide (GO) as support material, upconversion nanoparticles (UCNPs) as fluorescence signal reporter, N-isopropyl acrylamide (NIPAAM) and 4-vinylphenylboronic acid (VPBA) as functional monomers. The structure and component of smart sensing material was investigated by transmission electron microscopy (TEM), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopic (XPS) and Fourier transform infrared (FTIR), respectively. These results illustrated the smart sensing material was prepared successfully. The recognition characterizations of smart sensing material were evaluated, and results showed that the fluorescence intensity of smart sensing material was reduced gradually, as the concentration of protein increased, and the smart sensing material showed selective recognition for HRP among other proteins. Furthermore, the recognition ability of the smart sensing material for glycoprotein was regulated by controlling the pH value and temperature. Therefore, this strategy opens up new way to construct smart material for detection of glycoprotein. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2013-10-01

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

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

Optical Fluorescence Microscopy for Spatially Characterizing Electron Transfer across a Solid-Liquid Interface on Heterogeneous Electrodes.

PubMed

Choudhary, Eric; Velmurugan, Jeyavel; Marr, James M; Liddle, James A; Szalai, Veronika

2016-01-01

Heterogeneous catalytic materials and electrodes are used for (electro)chemical transformations, including those important for energy storage and utilization. 1, 2 Due to the heterogeneous nature of these materials, activity measurements with sufficient spatial resolution are needed to obtain structure/activity correlations across the different surface features (exposed facets, step edges, lattice defects, grain boundaries, etc.). These measurements will help lead to an understanding of the underlying reaction mechanisms and enable engineering of more active materials. Because (electro)catalytic surfaces restructure with changing environments, 1 it is important to perform measurements in operando . Sub-diffraction fluorescence microscopy is well suited for these requirements because it can operate in solution with resolution down to a few nm. We have applied sub-diffraction fluorescence microscopy to a thin cell containing an electrocatalyst and a solution containing the redox sensitive dye p-aminophenyl fluorescein to characterize reaction at the solid-liquid interface. Our chosen dye switches between a nonfluorescent reduced state and a one-electron oxidized bright state, a process that occurs at the electrode surface. This scheme is used to investigate the activity differences on the surface of polycrystalline Pt, in particular to differentiate reactivity at grain faces and grain boundaries. Ultimately, this method will be extended to study other dye systems and electrode materials.

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

Solid state protein monolayers: Morphological, conformational, and functional properties

NASA Astrophysics Data System (ADS)

Pompa, P. P.; Biasco, A.; Frascerra, V.; Calabi, F.; Cingolani, R.; Rinaldi, R.; Verbeet, M. Ph.; de Waal, E.; Canters, G. W.

2004-12-01

We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.

Effects of heat/citric acid reprocessing on high-flux polysulfone dialyzers.

PubMed

Cornelius, Rena M; McClung, W Glenn; Richardson, Robert M A; Estridge, Charles; Plaskos, Nicholas; Yip, Christopher M; Brash, John L

2002-01-01

The surface features, morphology, and tensile properties of fibers obtained from pristine, reprocessed, and reused Fresenius Polysulfone High-Flux (Hemoflow F80A) hemodialyzers have been studied. Scanning electron microscopy of the dialyzer fibers revealed a dense skin layer on the inner surface of the membrane and a relatively thick porous layer on the outer surface. Transmission electron microscopy and atomic force microscopy showed an alteration in membrane morphology due to reprocessing and reuse, or to a deposition of blood-borne material on the membrane that is not removed with reprocessing. Fluorescent microscopy images also showed that a fluorescent material not removed by heat/citric acid reprocessing builds up with continued use of the dialyzers. The tensile properties of the dialyzer fibers were not affected by the heat/citric acid reprocessing procedure. The protein layers formed on pristine and reused hemodialyzer membranes during clinical use were also studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. A considerable amount of protein was found on the blood side of single and multiple use dialyzers. Proteins adsorbed on the dialysate side of the membrane were predominantly in the molecular weight region below 30 kDa. Little protein was detected on the membranes of reprocessed hemodialyzers.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Characterization of a fluorescent hydrogel synthesized using chitosan, polyvinyl alcohol and 9-anthraldehyde for the selective detection and discrimination of trace Fe3+ and Fe2+ in water for live-cell imaging.

PubMed

Maity, Santu; Parshi, Nira; Prodhan, Chandraday; Chaudhuri, Keya; Ganguly, Jhuma

2018-08-01

A three-dimensional fluorescent hydrogel based on chitosan, polyvinyl alcohol and 9-anthraldehyde (ChPA) has been successfully designed and synthesized for the selective detection and discrimination of Fe 3+ and Fe 2+ in aqueous environment. The unique characteristics of ChPA has been confirmed by the Fourier-transform infrared spectroscopy (FTIR), rheological measurement, scanning electron microscopy (SEM), thermogravimetry and differential thermogravimetry (TG-DTG), ultraviolet-visible spectroscopy (UV-vis), fluorescence studies, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDX), x-ray diffraction (XRD) and dynamic light scattering (DLS). The emission intensity at 516 nm of the hydrogel has been enhanced remarkably with the addition of Fe 3+ due to the inhibition of the photoinduced electron transfer (PET) process. However, it gets strongly quenched in the case of Fe 2+ owing to chelation enhanced quenching (CHEQ). The probe (ChPA) causes no significant change in the fluorescence and becomes highly specific and sensitive towards Fe 3+ and Fe 2+ compared to other interfering heavy and transition metal ions (HTM). The detection limits of the sensor for the Fe 3+ and Fe 2+ are 0.124 nM and 0.138 nM, respectively. The probe is also promising as a selective sensor for the Fe 3+ and Fe 2+ in the fluorescence imaging of living cells. Thus, such a probe opens up new opportunities to improve the chitosan based fluorescent chemosensor having biocompatibility, biodegradability, sufficient thermal stability and stability in a wide pH range. Copyright © 2018 Elsevier Ltd. All rights reserved.

Photoinduced electron transfer of oxazine 1/TiO2 nanoparticles at single molecule level by using confocal fluorescence microscopy.

PubMed

Chen, Yi-Ju; Tzeng, Hsin-Yu; Fan, Hsiu-Fang; Chen, Ming-Shiang; Huang, Jer-Shing; Lin, King-Chuen

2010-06-01

Kinetics of photoinduced electron transfer (ET) from oxazine 1 dye to TiO(2) nanoparticles (NPs) surface is studied at a single molecule level by using confocal fluorescence microscopy. Upon irradiation with a pulsed laser at 630 nm, the fluorescence lifetimes sampled among 100 different dye molecules are determined to yield an average lifetime of 2.9 +/- 0.3 ns, which is close to the value of 3.0 +/- 0.6 ns measured on the bare coverslip. The lifetime proximity suggests that most interfacial electron transfer (IFET) processes for the current system are inefficient, probably caused by physisorption between dye and the TiO(2) film. However, there might exist some molecules which are quenched before fluorescing and fail to be detected. With the aid of autocorrelation analysis under a three-level energy system, the IFET kinetics of single dye molecules in the conduction band of TiO(2) NPs is evaluated to be (1.0 +/- 0.1) x 10(4) s(-1) averaged over 100 single molecules and the back ET rate constant is 4.7 +/- 0.9 s(-1). When a thicker TiO(2) film is substituted, the resultant kinetic data do not make a significant difference. The trend of IFET efficacy agrees with the method of fluorescence lifetime measurements. The obtained forward ET rate constants are about ten times smaller than the photovoltage response measured in an assembled dye-sensitized solar cell. The discrepancy is discussed. The inhomogeneous and fluctuation characters for the IFET process are attributed to microenvironment variation for each single molecule. The obtained ET rates are much slower than the fluorescence relaxation. Such a small ET quantum yield is yet feasibly detectable at a single molecule level.

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

Preparation of poly(methyl methacrylate) microcapsules by in situ polymerization on the surface of calcium carbonate particles.

PubMed

Sato, Katsuhiko; Nakajima, Tatsuya; Anzai, Jun-ichi

2012-12-01

Poly(methyl methacrylate) (PMMA) microcapsules were prepared by the in situ polymerization of methyl methacrylate (MMA) and N,N'-methylenebisacrylamide on the surface of calcium carbonate (CaCO(3)) particles, followed by the dissolution of the CaCO(3) core in ethylenediaminetetraacetic acid solution. The microcapsules were characterized using fluorescence microscopy, atomic force microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. The average sizes of the CaCO(3) particles and PMMA capsules were 3.8±0.6 and 4.0±0.6 μm, respectively. A copolymer consisting of MMA and rhodamine B-bearing MMA was also used to prepare microcapsules for fluorescent microscopy observations. Fluorescein isothiocyanate-labeled bovine serum albumin was enclosed in the PMMA microcapsules and its release properties were studied. Copyright © 2012 Elsevier Inc. All rights reserved.

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

PubMed Central

Vega, Israel A.; Castro-Vazquez, Alfredo

2015-01-01

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail. PMID:25893243

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney.

PubMed

Cueto, Juan A; Rodriguez, Cristian; Vega, Israel A; Castro-Vazquez, Alfredo

2015-01-01

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.

Morphology and dispersion of FeCo alloy nanoparticles dispersed in a matrix of IR pyrolized polyvinyl alcohol

NASA Astrophysics Data System (ADS)

Vasilev, A. A.; Dzidziguri, E. L.; Muratov, D. G.; Zhilyaeva, N. A.; Efimov, M. N.; Karpacheva, G. P.

2018-04-01

Metal-carbon nanocomposites consisting of FeCo alloy nanoparticles dispersed in a carbon matrix were synthesized by the thermal decomposition method of a precursor based on polyvinyl alcohol and metals salts. The synthesized powders were investigated by X-ray diffraction (XRD), X-ray fluorescent spectrometry (XRFS), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Surface characteristics of materials were measured by BET-method. The morphology and dispersity of metal nanoparticles were studied depending on the metals ratio in the composite.

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow.

PubMed

Barendrecht, Arjan D; Verhoef, Johan J F; Pignatelli, Silvia; Pasterkamp, Gerard; Heijnen, Harry F G; Maas, Coen

2017-07-10

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions. A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Spectroscopic study of Pbs nano-structured layer prepared by Pld utilized as a Hall-effect magnetic sensor

NASA Astrophysics Data System (ADS)

Atwa, D. M.; Aboulfotoh, N.; El-magd, A. Abo; Badr, Y.

2013-10-01

Lead sulfide (PbS) nano-structured films have been grown on quartz substrates using PLD technique. The deposited films were characterized by several structural techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Selected-area electron diffraction patterns (SAED). The results prove the formation of cubic phase of PbS nanocrystals. Elemental analysis of the deposited films compared to the bulk target was obtained via laser induced fluorescence of the produced plasma particles and the energy dispersive X-ray "EDX" technique. The Hall coefficient measurements indicate an efficient performance of the deposited films as a magnetic sensor.

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.

Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

NASA Astrophysics Data System (ADS)

Skala, Melissa Caroline

2007-12-01

Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology.

Pulse energy dependence of subcellular dissection by femtosecond laser pulses

NASA Technical Reports Server (NTRS)

Heisterkamp, A.; Maxwell, I. Z.; Mazur, E.; Underwood, J. M.; Nickerson, J. A.; Kumar, S.; Ingber, D. E.

2005-01-01

Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 micrometer away. c2005 Optical Society of America.

DMD-based LED-illumination super-resolution and optical sectioning microscopy.

PubMed

Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×10(7) pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

DMD-based LED-illumination Super-resolution and optical sectioning microscopy

PubMed Central

Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens. PMID:23346373

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.

Preparation and Characterization of Fluorescent SiO2 Microspheres

NASA Astrophysics Data System (ADS)

Xu, Cui; Zhang, Hao; Guan, Ruifang

2018-01-01

Fluorescent compound without typical fluorophores was synthesized with citric acid (CA) and aminopropyltriethoxysilane (APTS) firstly, and then it was grafted to the surface of the prepared SiO2 microspheres by chemical reaction. The fluorescent SiO2 microspheres with good fluorescent properties were obtained by optimizing the reaction conditions. And the morphology and structure of the fluorescent SiO2 microspheres have been characterized by scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. The results showed that the preparation of fluorescent SiO2 microspheres have good monodispersity and narrow particle size distribution. Moreover, the fluorescent SiO2 microspheres can be applied to detect Fe3+ in aqueous solution, prepare fluorescent SiO2 rubber, and have potential to be applied in the fluorescent labeling and fingerprint appearing technique fields.

Methods of chemical and phase composition analysis of gallstones

NASA Astrophysics Data System (ADS)

Suvorova, E. I.; Pantushev, V. V.; Voloshin, A. E.

2017-11-01

This review presents the instrumental methods used for chemical and phase composition investigation of gallstones. A great body of data has been collected in the literature on the presence of elements and their concentrations, obtained by fluorescence microscopy, X-ray fluorescence spectroscopy, neutron activation analysis, proton (particle) induced X-ray emission, atomic absorption spectroscopy, high-resolution gamma-ray spectrometry, electron paramagnetic resonance. Structural methods—powder X-ray diffraction, infrared spectroscopy, Raman spectroscopy—provide information about organic and inorganic phases in gallstones. Stone morphology was studied at the macrolevel with optical microscopy. Results obtained by analytical scanning and transmission electron microscopy with X-ray energy dispersive spectrometry are discussed. The chemical composition and structure of gallstones determine the strategy of removing stone from the body and treatment of patients: surgery or dissolution in the body. Therefore one chapter of the review describes the potential of dissolution methods. Early diagnosis and appropriate treatment of the disease depend on the development of clinical methods for in vivo investigation, which gave grounds to present the main characteristics and potential of ultrasonography (ultrasound scanning), magnetic resonance imaging, and X-ray computed tomography.

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.

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

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

PubMed Central

Kopek, Benjamin G.; Paez-Segala, Maria G.; Shtengel, Gleb; Sochacki, Kem A.; Sun, Mei G.; Wang, Yalin; Xu, C. Shan; van Engelenburg, Schuyler B.; Taraska, Justin W.; Looger, Loren L.; Hess, Harald F.

2017-01-01

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology. PMID:28384138

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.

PubMed

Ding, Shi-You; Xu, Qi; Ali, Mursheda K; Baker, John O; Bayer, Edward A; Barak, Yoav; Lamed, Raphael; Sugiyama, Junji; Rumbles, Garry; Himmel, Michael E

2006-10-01

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.

Small molecule PZL318: forming fluorescent nanoparticles capable of tracing their interactions with cancer cells and activated platelets, slowing tumor growth and inhibiting thrombosis

PubMed Central

Li, Shan; Wang, Yuji; Wang, Feng; Wang, Yaonan; Zhang, Xiaoyi; Zhao, Ming; Feng, Qiqi; Wu, Jianhui; Zhao, Shurui; Wu, Wei; Peng, Shiqi

2015-01-01

Low selectivity of chemotherapy correlates with poor outcomes of cancer patients. To improve this issue, a novel agent, N-(1-[3-methoxycarbonyl-4-hydroxyphenyl]-β-carboline-3-carbonyl)-Trp-Lys-OBzl (PZL318), was reported here. The transmission electron microscopy, scanning electron microscopy, and atomic force microscopy images demonstrated that PZL318 can form nanoparticles. Fluorescent and confocal images visualized that PZL318 formed fluorescent nanoparticles capable of targeting cancer cells and tracing their interactions with cancer cells. In vitro, 40 μM of PZL318 inhibited the proliferation of tumorigenic cells, but not nontumorigenic cells. In vivo, 10 nmol/kg of PZL318 slowed the tumor growth of S180 mice and alleviated the thrombosis of ferric chloride-treated ICR mice, while 100 μmol/kg of PZL318 did not injure healthy mice and they exhibited no liver toxicity. By analyzing Fourier transform–mass spectrometry and rotating-frame Overhauser spectroscopy (ROESY) two-dimensional nuclear magnetic resonance spectra, the chemical mechanism of PZL318-forming trimers and nanoparticles was explored. By using mesoscale simulation, a nanoparticle of 3.01 nm in diameter was predicted containing 13 trimers. Scavenging free radicals, downregulating sP-selectin expression and intercalating toward DNA were correlated with the antitumor mechanism of PZL318. PMID:26345234

Neural plasticity explored by correlative two-photon and electron/SPIM microscopy

NASA Astrophysics Data System (ADS)

Allegra Mascaro, A. L.; Silvestri, L.; Costantini, I.; Sacconi, L.; Maco, B.; Knott, G. W.; Pavone, F. S.

2013-06-01

Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

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

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

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

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

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

Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres,more » and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.« less

Inhibition of Fibrillar Assemblies of l-Phenylalanine by Crown Ethers: A Potential Approach toward Phenylketonuria.

PubMed

Banik, Debasis; Dutta, Rupam; Banerjee, Pavel; Kundu, Sangita; Sarkar, Nilmoni

2016-08-11

In this article, our aim is to investigate the interaction of l-phenylalanine (l-Phe) fibrils with crown ethers (CEs). For this purpose, two different CEs (15-Crown-5 (15C5) and 18-Crown-6 (18C6)) were used. Interestingly, we have observed that both CEs have the ability to arrest fibril formation. However, 18C6 was found to be a better candidate compared to 15C5. Field emission scanning electron microscopy and fluorescence lifetime imaging microscopy were used to monitor the fibril-arresting kinetics of CEs. The arresting process was further confirmed by fluorescence correlation spectroscopy and nuclear magnetic resonance studies.

Freeze-Fracture Ultrastructure of Thylakoid Membranes in Chloroplasts from Manganese-Deficient Plants

PubMed Central

Simpson, David J.; Robinson, Simon P.

1984-01-01

Leaves from spinach (Spinacia oleracea L. cv Hybrid 102) plants grown in Mn-deficient nutrient solution were characterized by chlorosis, lowered chlorophyll a/b ratio and reduced electron transport. There were characteristic changes in room temperature fluorescence induction kinetics with increased initial yield (Fo) and decreased variable fluorescence (Fv). The fluorescence yield after the maximum fell rapidly to a level below Fo. The shape of the rise from Fo to the maximum was altered and the size of photosystem II units increased, as measured by half-rise time of Fv in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The Mn-deficient leaves were harvested before necrosis, when thin section electron microscopy revealed no disorganization of the thylakoid system. Thylakoid membranes were examined by freeze-fracture electron microscopy. The effect of Mn-deficiency was the specific loss of three-quarters of the particles from the endoplasmic fracture face of appressed thylakoids (EFs). Mn-deficient leaves were restored to near normal 2 days after application of exogenous Mn to the nutrient solution. It is concluded that the loss of most, but not all, functional photosystem II reaction centers from grana, with no alteration in light-harvesting complex or photosystem I, is responsible for the fluorescence and functional properties observed. The response of thylakoids to Mn deficiency shows that there is a fundamental difference in composition and function of stacked and unstacked endoplasmic fracture particles. The stacked endoplasmic fracture particle probably contains, in close association, the photosystem II reaction center and also the Mn-containing polypeptide, the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-binding protein, and all electron transport components in between. Images Fig. 3 Fig. 4 Fig. 5 PMID:16663491

Cultivating Fluorescent Flowers with Highly Luminescent Carbon Dots Fabricated by a Double Passivation Method.

PubMed

Han, Shuai; Chang, Tao; Zhao, Haiping; Du, Huanhuan; Liu, Shan; Wu, Baoshuang; Qin, Shenjun

2017-07-07

In this work, we present the fabrication of highly luminescent carbon dots (CDs) by a double passivation method with the assistance of Ca(OH)₂. In the reaction process, Ca 2+ protects the active functional groups from overconsumption during dehydration and carbonization, and the electron-withdrawing groups on the CD surface are converted to electron-donating groups by the hydroxyl ions. As a result, the fluorescence quantum yield of the CDs was found to increase with increasing Ca(OH)₂ content in the reaction process. A blue-shift optical spectrum of the CDs was also found with increasing Ca(OH)₂ content, which could be attributed to the increasing of the energy gaps for the CDs. The highly photoluminescent CDs obtained (quantum yield: 86%) were used to cultivate fluorescent carnations by a water culture method, while the results of fluorescence microscopy analysis indicated that the CDs had entered the plant tissue structure.

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

Nanowires formed by the co-assembly of a negatively charged low-molecular weight gelator and a zwitterionic polythiophene.

PubMed

Li, Feng; Palaniswamy, Ganesan; de Jong, Menno R; Aslund, Andreas; Konradsson, Peter; Marcelis, Antonius T M; Sudhölter, Ernst J R; Stuart, Martien A Cohen; Leermakers, Frans A M

2010-06-21

Conjugated organic nanowires have been prepared by co-assembling a carboxylate containing low-molecular weight gelator (LMWG) and an amino acid substituted polythiophene derivative (PTT). Upon introducing the zwitterionic polyelectrolyte PTT to a basic molecular solution of the organogelator, the negative charges on the LMWG are compensated by the positive charges of the PTT. As a result, nanowires form through co-assembly. These nanowires are visualized by both transmission electron microscopy (TEM) and atomic force microscopy (AFM). Depending on the concentration and ratio of the components these nanowires can be micrometers long. These measurements further suggest that the aggregates adopt a helical conformation. The morphology of these nanowires are studied with fluorescent confocal laser scanning microscopy (CLSM). The interactions between LMWG and PTT are characterized by steady-state and time-resolved fluorescence spectroscopy studies. The steady-state spectra indicate that the backbone of the PTT adopts a more planar and more aggregated conformation when interacting with LMWG. The time- resolved fluorescence decay studies confirm this interpretation.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

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

Swulius, Matthew T.; Chen, Songye; Jane Ding, H.

2011-04-22

Highlights: {yields} No long helical filaments are seen near or along rod-shaped bacterial inner membranes by electron cryo-tomography. {yields} Electron cryo-tomography has the resolution to detect single filaments in vivo. -- Abstract: How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm)more » helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.« less

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

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Optical detection of λ-cyhalothrin by core-shell fluorescent molecularly imprinted polymers in Chinese spirits.

PubMed

Wang, Jixiang; Gao, Lin; Han, Donglai; Pan, Jianming; Qiu, Hao; Li, Hongji; Wei, Xiao; Dai, Jiangdong; Yang, Jinghai; Yao, Hui; Yan, Yongsheng

2015-03-11

In this study, fluorescent molecularly imprinted polymers (FMIPs), which were for the selective recognition and fluorescence detection of λ-cyhalothrin (LC), were synthesized via fluorescein 5(6)-isothiocyanate (FITC) and 3-aminopropyltriethoxysilane (APTS)/SiO2 particles. The SiO2@FITC-APTS@MIPs were characterized by Fourier transform infrared (FT-IR), UV-vis spectrophotometer (UV-vis), fluorescence spectrophotometer, thermogravimetric analysis (TGA), confocal laser scanning microscope (CLSM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The as-synthesized SiO2@FITC-APTS@MIPs with an imprinted polymer film (thickness was about 100 nm) was demonstrated to be spherically shaped and had good monodispersity, high fluorescence intensity, and good selective recognition. Using fluorescence quenching as the detection tool, the largest fluorescence quenching efficiency (F0/F - 1) of SiO2@FITC-APTS@MIPs is close to 2.5 when the concentration of the LC is 1.0 μM L(-1). In addition, a linear relationship (F0/F - 1= 0.0162C + 0.0272) could be obtained covering a wide concentration range of 0-60 nM L(-1) with a correlation coefficient of 0.9968 described by the Stern-Volmer equation. Moreover, the limit of detection (LOD) of the SiO2@FITC-APTS@MIPs was 9.17 nM L(-1). The experiment results of practical detection revealed that the SiO2@FITC-APTS@MIPs as an attractive recognition element was satisfactory for the determination of LC in Chinese spirits. Therefore, this study demonstrated the potential of SiO2@FITC-APTS@MIPs for the recognition and detection of LC in food.

Coherent anti-Stokes Raman scattering microscopy of single nanodiamonds

NASA Astrophysics Data System (ADS)

Pope, Iestyn; Payne, Lukas; Zoriniants, George; Thomas, Evan; Williams, Oliver; Watson, Peter; Langbein, Wolfgang; Borri, Paola

2014-11-01

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug-delivery vehicles and contrast agents in vivo. In the quest for superior photostability and biocompatibility, nanodiamonds are considered one of the best choices due to their unique structural, chemical, mechanical and optical properties. So far, mainly fluorescent nanodiamonds have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centres with stable optical properties. Here, we show that single non-fluorescing nanodiamonds exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp3 vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and nanodiamond size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of nanodiamonds internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively.

Coherent anti-Stokes Raman scattering microscopy of single nanodiamonds

PubMed Central

Pope, Iestyn; Payne, Lukas; Zoriniants, George; Thomas, Evan; Williams, Oliver; Watson, Peter; Langbein, Wolfgang; Borri, Paola

2016-01-01

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug delivery vehicles, and contrast agents in vivo. In the quest for superior photostability and bio-compatibility, nanodiamonds (NDs) are considered one of the best choices due to their unique structural, chemical, mechanical, and optical properties. So far, mainly fluorescent NDs have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centers with stable optical properties. Here, we show that single non-fluorescing NDs exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp3 vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and ND size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of NDs internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively. PMID:25305746

Coherent anti-Stokes Raman scattering microscopy of single nanodiamonds.

PubMed

Pope, Iestyn; Payne, Lukas; Zoriniants, George; Thomas, Evan; Williams, Oliver; Watson, Peter; Langbein, Wolfgang; Borri, Paola

2014-11-01

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug-delivery vehicles and contrast agents in vivo. In the quest for superior photostability and biocompatibility, nanodiamonds are considered one of the best choices due to their unique structural, chemical, mechanical and optical properties. So far, mainly fluorescent nanodiamonds have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centres with stable optical properties. Here, we show that single non-fluorescing nanodiamonds exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp(3) vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and nanodiamond size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of nanodiamonds internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively.

Ultratrace Detection of Nitroaromatics: Picric Acid Responsive Aggregation/Disaggregation of Self-Assembled p-Terphenylbenzimidazolium-Based Molecular Baskets.

PubMed

Sandhu, Sana; Kumar, Rahul; Singh, Prabhpreet; Mahajan, Aman; Kaur, Manmeet; Kumar, Subodh

2015-05-20

1-(p-Terphenyl)-benzimidazolium (TRIPOD-TP) molecules undergo self-assembly to form rodlike structures in aqueous medium, as shown by field-emission scanning electron microscopy, transmission electron microscopy, and dynamic light scattering studies. Upon gradual addition of picric acid (PA), these aggregates undergo an aggregation/disaggregation process to complex morphological structures (10(-12)-10(-10) M PA) and spherical aggregates (10(-9)-10(-8) M PA). These spherical aggregates undergo further dissolution to well-dispersed spheres between 10(-7)-10(-6) M PA. During fluorescence studies, these aggregates demonstrate superamplified fluorescence quenching (>97%) in the presence of 10(-5) to 0.2 equiv of the probe concentration, an unprecedented process with PA. The lowest detection limits by solution of TRIPOD-TP are 5 × 10(-13) PA, 50 × 10(-12) M 2,4-dinitrophenol, 200 × 10(-12) M 2,4,6-trinitrotoluene, and 1 nM 1-chloro-2,4-dinitrobenzene. Paper strips dipped in the solution of TRIPOD-TP demonstrate quantitative fluorescence quenching between 10(-17) and 10(-6) M PA using front-surface steady state studies and can measure as low as 2.29 × 10(-20) g/cm(2) PA.

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

PubMed

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

2014-01-01

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

CHARACTERIZATION OF PARTICULATE MATTER FROM PHOENIX, ARIZONA, USING RAY FLUORESCENCE AND COMPUTER-CONTROLLED SCANNING ELECTRON MICROSCOPY

EPA Science Inventory

Numerous epidemiological studies have found associations between airborne particulate matter measured at community monitors and increased mortality and morbidity. Chemical and physical characteristics of particulate matter (e.g., elemental composition, size) and source identifi...

Tracing nanoparticles and photosensitizing molecules at transmission electron microscopy by diaminobenzidine photo-oxidation.

PubMed

Malatesta, M; Pellicciari, C; Cisterna, B; Costanzo, M; Galimberti, V; Biggiogera, M; Zancanaro, C

2014-04-01

During the last three decades, diaminobenzidine photo-oxidation has been applied in a variety of studies to correlate light and electron microscopy. Actually, when a fluorophore is excited by light, it can induce the oxidation of diaminobenzidine into an electron-dense osmiophilic product, which precipitates in close proximity to the fluorophore, thereby allowing its ultrastructural detection. This method has very recently been developed for two innovative applications: tracking the fate of fluorescently labeled nanoparticles in single cells, and detecting the subcellular location of photo-active molecules suitable for photodynamic therapy. These studies established that the cytochemical procedures exploiting diaminobenzidine photo-oxidation represent a reliable tool for detecting, inside the cells, with high sensitivity fluorescing molecules. These procedures are trustworthy even if the fluorescing molecules are present in very low amounts, either inside membrane-bounded organelles, or at the surface of the plasma membrane, or free in the cytosol. In particular, diaminobenzidine photo-oxidation allowed elucidating the mechanisms responsible for nanoparticles internalization in neuronal cells and for their escape from lysosomal degradation. As for the photo-active molecules, their subcellular distribution at the ultrastructural level provided direct evidence for the lethal multiorganelle photo-damage occurring after cell photo-sensitization. In addition, DAB photo-oxidized samples are suitable for the ultrastructural detection of organelle-specific molecules by post-embedding gold immunolabeling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Attaching quantum dots to HER2 specific phage antibodies

NASA Astrophysics Data System (ADS)

Chu, Viet Ha; Nghiem, Thi Ha Lien; Huyen La, Thi; Dieu Thuy Ung, Thi; Huan Le, Quang; Thuan Tong, Kim; Liem Nguyen, Quang; Nhung Tran, Hong

2010-06-01

This work presents the results of the attachment of Qdot 655 ITKTM amino (PEG) quantum dots (QDs) (Invitrogen) and CdTe QDs (provided by Institute of Materials Science, VAST) to HER2 (Human Epidermal growth factor Receptor 2) specific phage antibodies (Abs) (provided by Institute of Biotechnology, VAST) in solution. The QDs were attached to the phage display specific HER2 Abs to form a complex QD-Ab. The QDs and complex QD-Ab were characterized by UV-VIS spectroscopy, transmission electron microscopy (TEM) and fluorescence microscopy. The fluorescence images show the QDs conjugated to the phage. Due to the QDs attaching to the surface, the phage dimensions were amplified, so its shape could be observed by optical microscopy. The complex QD-Ab was stable and lasted for a month. The results illustrate the value of the HER2 phage-QD complex as a cancer detection platform.

Biomedical Applications of Nanodiamonds: An Overview.

PubMed

Passeri, D; Rinaldi, F; Ingallina, C; Carafa, M; Rossi, M; Terranova, M L; Marianecci, C

2015-02-01

Nanodiamonds are a novel class of nanomaterials which have raised much attention for application in biomedical field, as they combine the possibility of being produced on large scale using relatively inexpensive synthetic processes, of being fluorescent as a consequence of the presence of nitrogen vacancies, of having their surfaces functionalized, and of having good biocompatibility. Among other applications, we mainly focus on drug delivery, including cell interaction, targeting, cancer therapy, gene and protein delivery. In addition, nanodiamonds for bone and dental implants and for antibacterial use is discussed. Techniques for detection and imaging of nanodiamonds in biological tissues are also reviewed, including electron microscopy, fluorescence microscopy, Raman mapping, atomic force microscopy, thermal imaging, magnetic resonance imaging, and positron emission tomography, either in vitro, in vivo, or ex vivo. Toxicological aspects related to the use of nanodiamonds are also discussed. Finally, patents, preclinical and clinical trials based on the use of nanodiamonds for biomedical applications are reviewed.

Internalization of Carbon Nano-onions by Hippocampal Cells Preserves Neuronal Circuit Function and Recognition Memory.

PubMed

Trusel, Massimo; Baldrighi, Michele; Marotta, Roberto; Gatto, Francesca; Pesce, Mattia; Frasconi, Marco; Catelani, Tiziano; Papaleo, Francesco; Pompa, Pier Paolo; Tonini, Raffaella; Giordani, Silvia

2018-05-23

One area where nanomedicine may offer superior performances and efficacy compared to current strategies is in the diagnosis and treatment of central nervous system (CNS) diseases. However, the application of nanomaterials in such complex arenas is still in its infancy and an optimal vector for the therapy of CNS diseases has not been identified. Graphitic carbon nano-onions (CNOs) represent a class of carbon nanomaterials that shows promising potential for biomedical purposes. To probe the possible applications of graphitic CNOs as a platform for therapeutic and diagnostic interventions on CNS diseases, fluorescently labeled CNOs were stereotaxically injected in vivo in mice hippocampus. Their diffusion within brain tissues and their cellular localization were analyzed ex vivo by confocal microscopy, electron microscopy, and correlative light-electron microscopy techniques. The subsequent fluorescent staining of hippocampal cells populations indicates they efficiently internalize the nanomaterial. Furthermore, the inflammatory potential of the CNOs injection was found comparable to sterile vehicle infusion, and it did not result in manifest neurophysiological and behavioral alterations of hippocampal-mediated functions. These results clearly demonstrate that CNOs can interface effectively with several cell types, which encourages further their development as possible brain disease-targeted diagnostics or therapeutics nanocarriers.

[Construction of Lactobacillus rhamnosus GG particles surface display system].

PubMed

Su, Runyu; Nie, Boyao; Yuan, Shengling; Tao, Haoxia; Liu, Chunjie; Yang, Bailiang; Wang, Yanchun

2017-01-25

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

PubMed

Rizzo, N W; Duncan, K E; Bourett, T M; Howard, R J

2016-08-01

We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. © 2015 The Author. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of the Royal Microscopical Society.

New methods allowing the detection of protein aggregates

PubMed Central

Demeule, Barthélemy; Palais, Caroline; Machaidze, Gia; Gurny, Robert

2009-01-01

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin®) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates. PMID:20061815

D-penicillamine-templated copper nanoparticles via ascorbic acid reduction as a mercury ion sensor.

PubMed

Lin, Shu Min; Geng, Shuo; Li, Na; Li, Nian Bing; Luo, Hong Qun

2016-05-01

Mercury ion is one of the most hazardous metal pollutants that can cause deleterious effects on human health and the environment even at low concentrations. It is necessary to develop new mercury detection methods with high sensitivity, specificity and rapidity. In this study, a novel and green strategy for synthesizing D-penicillamine-capped copper nanoparticles (DPA-CuNPs) was successfully established by a chemical reduction method, in which D-penicillamine and ascorbic acid were used as stabilizing agent and reducing agent, respectively. The as-prepared DPA-CuNPs showed strong red fluorescence and had a large Stoke's shift (270nm). Scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectrophotometry were utilized to elucidate the possible fluorescence mechanism, which could be aggregation-induced emission effect. Based on the phenomenon that trace mercury ion can disperse the aggregated DPA-CuNPs, resulting in great fluorescence quench of the system, a sensitive and selective assay for mercury ion in aqueous solution with the DPA-CuNPs was developed. Under optimum conditions, this assay can be applied to the quantification of Hg(2+) in the 1.0-30μM concentration range and the detection limit (3σ/slope) is 32nM. The method was successfully applied to determine Hg(2+) in real water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Three-dimensional textures and defects of soft material layering revealed by thermal sublimation.

PubMed

Yoon, Dong Ki; Kim, Yun Ho; Kim, Dae Seok; Oh, Seong Dae; Smalyukh, Ivan I; Clark, Noel A; Jung, Hee-Tae

2013-11-26

Layering is found and exploited in a variety of soft material systems, ranging from complex macromolecular self-assemblies to block copolymer and small-molecule liquid crystals. Because the control of layer structure is required for applications and characterization, and because defects reveal key features of the symmetries of layered phases, a variety of techniques have been developed for the study of soft-layer structure and defects, including X-ray diffraction and visualization using optical transmission and fluorescence confocal polarizing microscopy, atomic force microscopy, and SEM and transmission electron microscopy, including freeze-fracture transmission electron microscopy. Here, it is shown that thermal sublimation can be usefully combined with such techniques to enable visualization of the 3D structure of soft materials. Sequential sublimation removes material in a stepwise fashion, leaving a remnant layer structure largely unchanged and viewable using SEM, as demonstrated here using a lamellar smectic liquid crystal.

Quantitative Connection Between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryo-EM and smFRET Investigations of the Ribosome

PubMed Central

Frank, Joachim; Gonzalez, Ruben L.

2015-01-01

At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describes transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy and single-molecule fluorescence resonance energy transfer studies of the bacterial ribosomal pretranslocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pretranslocation complex, which are observed in a cryogenic electron microscopy study, may not be observed in several single-molecule fluorescence resonance energy transfer studies. PMID:25785884

Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome.

PubMed

Thompson, Colin D Kinz; Sharma, Ajeet K; Frank, Joachim; Gonzalez, Ruben L; Chowdhury, Debashish

2015-08-27

At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describe transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies of the bacterial ribosomal pre-translocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pre-translocation complex, which are observed in a cryo-EM study, may not be observed in several smFRET studies.

Functionalized Buckyballs for Visualizing Microbial Species in Different States and Environments

DOE PAGES

Cheng, Qingsu; Aravind, Ashwin; Buckley, Matthew; ...

2015-09-08

To date, in situ visualization of microbial density has remained an open problem. Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil. These molecular probes are validated via multi-scale imaging, to show association with microorganisms via fluorescence microscopy, positive cellular uptake via electron microscopy, and non-specific binding to the substrates through a combination of fluorescence and autoradiography imaging. In conclusion, we also demonstrate that cysteine-functionalized C60- pyrrolidine tris acid canmore » differentiate live and dead microorganisms.« less

Methods to Assess Mitochondrial Morphology in Mammalian Cells Mounting Autophagic or Mitophagic Responses.

PubMed

Marchi, S; Bonora, M; Patergnani, S; Giorgi, C; Pinton, P

2017-01-01

It is widely acknowledged that mitochondria are highly active structures that rapidly respond to cellular and environmental perturbations by changing their shape, number, and distribution. Mitochondrial remodeling is a key component of diverse biological processes, ranging from cell cycle progression to autophagy. In this chapter, we describe different methodologies for the morphological study of the mitochondrial network. Instructions are given for the preparation of samples for fluorescent microscopy, based on genetically encoded strategies or the employment of synthetic fluorescent dyes. We also propose detailed protocols to analyze mitochondrial morphometric parameters from both three-dimensional and bidimensional datasets. Finally, we describe a protocol for the visualization and quantification of mitochondrial structures through electron microscopy. © 2017 Elsevier Inc. All rights reserved.

[Preparation of polyelectrolyte microcapsules contained gold nanoparticles].

PubMed

Sun, Ya-jie; Zhu, Jia-bi; Zheng, Chun-li

2010-03-01

In this work, polyelectrolyte microcapsules containing gold nanoparticles were prepared via layer by layer assembly. Gold nanoparticles and poly (allyamine hydrochloride) (PAH) were coated on the CaCO3 microparticles. And then EDTA was used to remove the CaCO3 core. Scanning electron microscopy (SEM) was used to characterize the surface of microcapsules. SEM images indicate that the microcapsules and the polyelectrolyte multilayer were deposited on the surface of CaCO3 microparticles. FITC-bovine serum albumin (FITC-BSA, 2 mg) was incorporated in the CaCO3 microparticles by co-precipitation. Fluorescence microscopy was used to observe the fluorescence intensity of microcapsules. The encapsulation efficiency was (34.31 +/- 2.44) %. The drug loading was (43.75 +/- 3.12) mg g(-1).

Optical properties of hydrothermally synthesized TGA-capped CdS nanoparticles: controlling crystalline size and phase

NASA Astrophysics Data System (ADS)

Tavakoli Banizi, Zoha; Seifi, Majid

2017-10-01

TGA-capped CdS nanoparticles were obtained in the presence of thioglycolic acid (TGA) as capping agent via a facile hydrothermal method at relatively low temperature and over a short duration. As-synthesized TGA-capped CdS nanoparticles were characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectra, photoluminescence spectroscopy, Ultraviolet-visible spectroscopy and energy-dispersive x-ray spectroscopy. The products had spherical shapes, although their crystalline size and phase was dependent on temperature and time of the reaction. Photoluminescence spectra showed that the fluorescence intensity decreased when increasing the reaction time and temperature.

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

PubMed

Squire, Kenneth; Kong, Xianming; LeDuff, Paul; Rorrer, Gregory L; Wang, Alan X

2018-05-16

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 -16 M (14 fg/mL) and 10 -15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing. Scanning electron microscope image of biosilica diatom frustule that enables significant enhancement of fluorescence spectroscopy and fluorescence image. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Morphological study of fluorescent carbon Nanoparticles (F-CNPs) from ground coffee waste soot oxidation by diluted acid

NASA Astrophysics Data System (ADS)

Gea, S.; Tjandra, S.; Joshua, J.; Wirjosentono, B.

2018-02-01

Coffee ground waste utilization for fluorescent carbon nanoparticles (F-CNPs) through soot oxidation with diluted HNO3 has been conducted. Soot was obtained through three different treatments to coffee ground waste; which was burned in furnaceat 550°C and 650°C and directly burned in a heat-proofcontainer. Then they were analyzed morphologically with Scanning Electron Microscope (SEM) instrument. Soot from direct burning indicated the optimum result where it has denser pores compared to other two soots. Soot obtained from direct burning was refluxed in diluted HNO3 for 12 hours to perform the oxidation. Yellowish brown supernatant was later observed which lead to green fluorescent under the UV light. F-CNPs characterization was done in Transmission Electron Microscopy, which showed that 7.4-23.4 nm of particle size were distributed.

Next-Generation Theranostic Agents Based on Polyelectrolyte Microcapsules Encoded with Semiconductor Nanocrystals: Development and Functional Characterization

NASA Astrophysics Data System (ADS)

Nifontova, Galina; Zvaigzne, Maria; Baryshnikova, Maria; Korostylev, Evgeny; Ramos-Gomes, Fernanda; Alves, Frauke; Nabiev, Igor; Sukhanova, Alyona

2018-01-01

Fabrication of polyelectrolyte microcapsules and their use as carriers of drugs, fluorescent labels, and metal nanoparticles is a promising approach to designing theranostic agents. Semiconductor quantum dots (QDs) are characterized by extremely high brightness and photostability that make them attractive fluorescent labels for visualization of intracellular penetration and delivery of such microcapsules. Here, we describe an approach to design, fabricate, and characterize physico-chemical and functional properties of polyelectrolyte microcapsules encoded with water-solubilized and stabilized with three-functional polyethylene glycol derivatives core/shell QDs. Developed microcapsules were characterized by dynamic light scattering, electrophoretic mobility, scanning electronic microscopy, and fluorescence and confocal microscopy approaches, providing exact data on their size distribution, surface charge, morphological, and optical characteristics. The fluorescence lifetimes of the QD-encoded microcapsules were also measured, and their dependence on time after preparation of the microcapsules was evaluated. The optimal content of QDs used for encoding procedure providing the optimal fluorescence properties of the encoded microcapsules was determined. Finally, the intracellular microcapsule uptake by murine macrophages was demonstrated, thus confirming the possibility of efficient use of developed system for live cell imaging and visualization of microcapsule transportation and delivery within the living cells.

Non-covalent attachment of silver nanoclusters onto single-walled carbon nanotubes with human serum albumin as linking molecule

NASA Astrophysics Data System (ADS)

Rodríguez-Galván, Andrés; Heredia, Alejandro; Amelines-Sarria, Oscar; Rivera, Margarita; Medina, Luis A.; Basiuk, Vladimir A.

2015-03-01

The attachment of silver nanoclusters (AgNCs) onto single-walled carbon nanotubes (SWNTs) for the formation of integrated fluorescence sites has attracted much attention due their potential applications as biological probes and nanovectors in theragnosis. Here, we report the preparation through assembly of fluorescent quasi 1-D nanomaterial based on SWNTs and silver nanoclusters (AgNCs) non-covalently attached to human serum albumin as biological linker. The fluorescent SWNT-AgNCs-HSA conjugates were characterized by atomic force microscopy, high-resolution transmission electron microscopy (HRTEM), high angle annular dark field scanning TEM (HAADF-STEM), fluorescent and UV-vis spectroscopy. The above techniques confirmed that AgNCs were non-covalently attached onto the external surface of SWNTs. In addition, it was observed that the modification did not affect the optical properties of the synthesized AgNCs since the absorption spectra and fluorescence under UV irradiation (λ = 365 nm) remain the same. The effect of the functionalized systems was tested on mammal red blood cells (RBCs) and it was found that their structural integrity was compromised by the conjugates, limiting their biological and medical applications.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature.

PubMed

Garstka, Maciej; Venema, Jan Henk; Rumak, Izabela; Gieczewska, Katarzyna; Rosiak, Malgorzata; Koziol-Lipinska, Joanna; Kierdaszuk, Borys; Vredenberg, Wim J; Mostowska, Agnieszka

2007-10-01

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements.

A space- and time-resolved single photon counting detector for fluorescence microscopy and spectroscopy

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2017-01-01

We have recently developed a wide-field photon-counting detector having high-temporal and high-spatial resolutions and capable of high-throughput (the H33D detector). Its design is based on a 25 mm diameter multi-alkali photocathode producing one photo electron per detected photon, which are then multiplied up to 107 times by a 3-microchannel plate stack. The resulting electron cloud is proximity focused on a cross delay line anode, which allows determining the incident photon position with high accuracy. The imaging and fluorescence lifetime measurement performances of the H33D detector installed on a standard epifluorescence microscope will be presented. We compare them to those of standard single-molecule detectors such as single-photon avalanche photodiode (SPAD) or electron-multiplying camera using model samples (fluorescent beads, quantum dots and live cells). Finally, we discuss the design and applications of future generation of H33D detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:29479130

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

PubMed

Fermie, Job; Liv, Nalan; Ten Brink, Corlinda; van Donselaar, Elly G; Müller, Wally H; Schieber, Nicole L; Schwab, Yannick; Gerritsen, Hans C; Klumperman, Judith

2018-05-01

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag.

PubMed

Swulius, Matthew T; Jensen, Grant J

2012-12-01

Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP(SW)) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, "patchy" localization patterns of MreB-RFP(SW), even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.

Fluorescent single-digit detonation nanodiamond for biomedical applications

NASA Astrophysics Data System (ADS)

Nunn, Nicholas; d’Amora, Marta; Prabhakar, Neeraj; Panich, Alexander M.; Froumin, Natalya; Torelli, Marco D.; Vlasov, Igor; Reineck, Philipp; Gibson, Brant; Rosenholm, Jessica M.; Giordani, Silvia; Shenderova, Olga

2018-07-01

Detonation nanodiamonds (DNDs) have emerged as promising candidates for a variety of biomedical applications, thanks to different physicochemical and biological properties, such as small size and reactive surfaces. In this study, we propose carbon dot decorated single digit (4–5 nm diameter) primary particles of detonation nanodiamond as promising fluorescent probes. Due to their intrinsic fluorescence originating from tiny (1–2 atomic layer thickness) carbonaceous structures on their surfaces, they exhibit brightness suitable for in vitro imaging. Moreover, this material offers a unique, cost effective alternative to sub-10 nm nanodiamonds containing fluorescent nitrogen-vacancy color centers, which have not yet been produced at large scale. In this paper, carbon dot decorated nanodiamonds are characterized by several analytical techniques. In addition, the efficient cellular uptake and fluorescence of these particles are observed in vitro on MDA-MD-231 breast cancer cells by means of confocal imaging. Finally, the in vivo biocompatibility of carbon dot decorated nanodiamonds is demonstrated in zebrafish during the development. Our results indicate the potential of single-digit detonation nanodiamonds as biocompatible fluorescent probes. This unique material will find application in correlative light and electron microscopy, where small sized NDs can be attached to antibodies to act as a suitable dual marker for intracellular correlative microscopy of biomolecules.

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

Augmented microscopy with near-infrared fluorescence detection

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-03-01

Near-infrared (NIR) fluorescence has become a frequently used intraoperative technique for image-guided surgical interventions. In procedures such as cerebral angiography, surgeons use the optical surgical microscope for the color view of the surgical field, and then switch to an electronic display for the NIR fluorescence images. However, the lack of stereoscopic, real-time, and on-site coregistration adds time and uncertainty to image-guided surgical procedures. To address these limitations, we developed the augmented microscope, whereby the electronically processed NIR fluorescence image is overlaid with the anatomical optical image in real-time within the optical path of the microscope. In vitro, the augmented microscope can detect and display indocyanine green (ICG) concentrations down to 94.5 nM, overlaid with the anatomical color image. We prepared polyacrylamide tissue phantoms with embedded polystyrene beads, yielding scattering properties similar to brain matter. In this model, 194 μM solution of ICG was detectable up to depths of 5 mm. ICG angiography was then performed in anesthetized rats. A dynamic process of ICG distribution in the vascular system overlaid with anatomical color images was observed and recorded. In summary, the augmented microscope demonstrates NIR fluorescence detection with superior real-time coregistration displayed within the ocular of the stereomicroscope. In comparison to other techniques, the augmented microscope retains full stereoscopic vision and optical controls including magnification and focus, camera capture, and multiuser access. Augmented microscopy may find application in surgeries where the use of traditional microscopes can be enhanced by contrast agents and image guided delivery of therapeutics, including oncology, neurosurgery, and ophthalmology.

Spiculogenesis in the siliceous sponge Lubomirskia baicalensis studied with fluorescent staining.

PubMed

Annenkov, Vadim V; Danilovtseva, Elena N

2016-04-01

Siliceous sponges are the most primitive multicellular animals whose skeleton consists of spicules - needle-like constructions from silicon dioxide surrounding organic axial filaments. Mechanisms of spicule formation have been intensively studied due to the high ecological importance of sponges and their interest to materials science. Light and electron microscopy are not appropriate enough to display the process from silicon-enriched cells to mature spicules because of composite structure of the sponge tissues. In this article, spiculogenesis in the siliceous sponge has been studied for the first time with the use of fluorescent microscopy. Fluorescent vital dye NBD-N2 was applied to stain growing siliceous structures in the sponge and primmorph cell system. The main stages of spicule growth in the fresh-water sponge Lubomirskia baicalensis (Pallas, 1773) were visualized: silicon accumulation in sclerocytes; formation of an organic filament protruding from the cell; further elongation of the filament and growth of the spicule in a spindle-like form with enlargement in the center; merger with new sclerocytes and formation of the mature spicule. Fluorescent microscopy combined with SEM allows us to overcome the virtual differentiation between intra- and extracellular mechanisms of spicule growth. The growing spicule can capture silicic acid from the extracellular space and merge with new silicon-enriched cells. Visualization of the growing spicules with the fluorescent dye allows us to monitor sponge viability in ecological or toxicological experiments and to apply genomic, proteomic and biochemical techniques. Copyright © 2016 Elsevier Inc. All rights reserved.

Magnetic removal of Entamoeba cysts from water using chitosan oligosaccharide-coated iron oxide nanoparticles

PubMed Central

Shukla, Sudeep; Arora, Vikas; Jadaun, Alka; Kumar, Jitender; Singh, Nishant; Jain, Vinod Kumar

2015-01-01

Amebiasis, a major health problem in developing countries, is the second most common cause of death due to parasitic infection. Amebiasis is usually transmitted by the ingestion of Entamoeba histolytica cysts through oral–fecal route. Herein, we report on the use of chitosan oligosaccharide-functionalized iron oxide nanoparticles for efficient capture and removal of pathogenic protozoan cysts under the influence of an external magnetic field. These nanoparticles were synthesized through a chemical synthesis process. The synthesized particles were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and zeta potential analysis. The particles were found to be well dispersed and uniform in size. The capture and removal of pathogenic cysts were demonstrated by fluorescent microscopy, transmission electron microscopy, and scanning electron microscopy (SEM). Three-dimensional modeling of various biochemical components of cyst walls, and thereafter, flexible docking studies demonstrate the probable interaction mechanism of nanoparticles with various components of E. histolytica cyst walls. Results of the present study suggest that E. histolytica cysts can be efficiently captured and removed from contaminated aqueous systems through the application of synthesized nanoparticles. PMID:26261417

Microscopy techniques in flavivirus research.

PubMed

Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

2014-04-01

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Micro-RNA detection based on fluorescence resonance energy transfer of DNA-carbon quantum dots probes.

PubMed

Khakbaz, Faeze; Mahani, Mohamad

2017-04-15

Carbon quantum dots have been proposed as an effective platform for miRNA detection. Carbon dots were synthesized by citric acid. The synthesized dots were characterized by dynamic light scattering, UV-Vis spectrophotometry, spectrofluorimetry, transmission electron microscopy and FT-IR spectrophotometry. The fluorescence quantum yield of the synthesized dots was determined using quinine sulfate as the standard. The FAM-labeled single stranded DNA, as sensing element, was adsorbed on dots by π-π interaction. The quenching of the dots fluorescence due to fluorescence resonance energy transfer (FRET) was used for mir 9-1 detection. In the presence of the complementary miRNA, the FRET did not take place and the fluorescence was recovered. Copyright © 2017 Elsevier Inc. All rights reserved.

Cultivating Fluorescent Flowers with Highly Luminescent Carbon Dots Fabricated by a Double Passivation Method

PubMed Central

Chang, Tao; Zhao, Haiping; Du, Huanhuan; Liu, Shan; Wu, Baoshuang; Qin, Shenjun

2017-01-01

In this work, we present the fabrication of highly luminescent carbon dots (CDs) by a double passivation method with the assistance of Ca(OH)2. In the reaction process, Ca2+ protects the active functional groups from overconsumption during dehydration and carbonization, and the electron-withdrawing groups on the CD surface are converted to electron-donating groups by the hydroxyl ions. As a result, the fluorescence quantum yield of the CDs was found to increase with increasing Ca(OH)2 content in the reaction process. A blue-shift optical spectrum of the CDs was also found with increasing Ca(OH)2 content, which could be attributed to the increasing of the energy gaps for the CDs. The highly photoluminescent CDs obtained (quantum yield: 86%) were used to cultivate fluorescent carnations by a water culture method, while the results of fluorescence microscopy analysis indicated that the CDs had entered the plant tissue structure. PMID:28686178

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

PubMed

Fu, Hongxia; Li, Yanru; Sun, Lingbo; He, Pan; Duan, Xinrui

2015-11-17

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

PubMed

Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

2012-12-01

We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

Self-consistent method for quantifying indium content from X-ray spectra of thick compound semiconductor specimens in a transmission electron microscope.

PubMed

Walther, T; Wang, X

2016-05-01

Based on Monte Carlo simulations of X-ray generation by fast electrons we calculate curves of effective sensitivity factors for analytical transmission electron microscopy based energy-dispersive X-ray spectroscopy including absorption and fluorescence effects, as a function of Ga K/L ratio for different indium and gallium containing compound semiconductors. For the case of InGaN alloy thin films we show that experimental spectra can thus be quantified without the need to measure specimen thickness or density, yielding self-consistent values for quantification with Ga K and Ga L lines. The effect of uncertainties in the detector efficiency are also shown to be reduced. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Label-free real-time imaging of myelination in the Xenopus laevis tadpole by in vivo stimulated Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Hu, Chun-Rui; Zhang, Delong; Slipchenko, Mikhail N.; Cheng, Ji-Xin; Hu, Bing

2014-08-01

The myelin sheath plays an important role as the axon in the functioning of the neural system, and myelin degradation is a hallmark pathology of multiple sclerosis and spinal cord injury. Electron microscopy, fluorescent microscopy, and magnetic resonance imaging are three major techniques used for myelin visualization. However, microscopic observation of myelin in living organisms remains a challenge. Using a newly developed stimulated Raman scattering microscopy approach, we report noninvasive, label-free, real-time in vivo imaging of myelination by a single-Schwann cell, maturation of a single node of Ranvier, and myelin degradation in the transparent body of the Xenopus laevis tadpole.

Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.

PubMed

Crossman, David J; Ruygrok, Peter N; Hou, Yu Feng; Soeller, Christian

2015-03-01

Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds.

PubMed

Kim, Min Jeong; Shin, Yong Cheol; Lee, Jong Ho; Jun, Seung Won; Kim, Chang-Seok; Lee, Yunki; Park, Jong-Chul; Lee, Soo-Hong; Park, Ki Dong; Han, Dong-Wook

2016-01-01

Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM. The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope. Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

Photochemical and photocatalytic evaluation of 1D titanate/TiO2 based nanomaterials

NASA Astrophysics Data System (ADS)

Conceição, D. S.; Ferreira, D. P.; Graça, C. A. L.; Júlio, M. F.; Ilharco, L. M.; Velosa, A. C.; Santos, P. F.; Vieira Ferreira, L. F.

2017-01-01

One-dimensional (1D) titanate based nanomaterials were synthesized following an alkaline hydrothermal approach of commercial TiO2 nanopowder. The morphological features of all materials were monitored by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and also Brunauer-Emmett-Teller (BET) technique. In addition the photochemical behaviour of these nanostructured materials were evaluated with the use of laser induced luminescence (LIL), ground-state diffuse reflectance (GSDR), and laser-flash photolysis in diffuse reflectance mode (DRLFP). The mixed titanate/TiO2 nanowires presented the least intense fluorescence spectra, suggesting the presence of surficial defects that can extend the lifetime of the excited charge carriers. A fluorescent 'rhodamine-like' dye was adsorbed onto different materials and examined via photoexcitation in the visible range to study the self-photosensitization mechanism. The presence of the radical cation of the dye and the degradation kinetics, when compared with a neutral substrate-cellulose, provided significant evidences regarding the photoactivity of the different materials. Regarding all the materials under study, the nanowires exhibited a strong photocatalytic efficiency, for the adsorbed fluorescent probe. The photocatalytic mechanism was also considered by studying the photodegradation capability of the titanate based materials in the presence of an herbicide, Amicarbazone, after ultraviolet (UVA) photoexcitation.

Processing of copper converter slag for metals reclamation: Part II: mineralogical study.

PubMed

Deng, Tong; Ling, Yunhan

2004-10-01

Chemical and mineralogical characterizations of a copper converter slag, and its products obtained by curing with strong sulphuric acid and leaching with hot water, were carried out using ore microscopy, scanning electronic microscopy with energy dispersive spectrometry, wave-length dispersive X-ray fluorescence spectrometry, X-ray diffractometry and chemical phase analysis, which provided necessary information to develop a new process for treating such slag and further understanding of the chemical and mineralogical changes in the process.

One-Pot Exfoliation of Graphite and Synthesis of Nanographene/Dimesitylporphyrin Hybrids

PubMed Central

Bernal, M. Mar; Pérez, Emilio M.

2015-01-01

A simple one-pot process to exfoliate graphite and synthesize nanographene-dimesitylporphyrin hybrids has been developed. Despite the bulky mesityl groups, which are expected to hinder the efficient π–π stacking between the porphyrin core and graphene, the liquid-phase exfoliation of graphite is significantly favored by the presence of the porphyrins. Metallation of the porphyrin further enhances this effect. The resulting graphene/porphyrin hybrids were characterized by spectroscopy (UV-visible, fluorescence, and Raman) and microscopy (STEM, scanning transmission electron microscopy). PMID:25984598

Synthesis of strongly fluorescent molybdenum disulfide nanosheets for cell-targeted labeling.

PubMed

Wang, Nan; Wei, Fang; Qi, Yuhang; Li, Hongxiang; Lu, Xin; Zhao, Guoqiang; Xu, Qun

2014-11-26

MoS2 nanosheets with polydispersity of the lateral dimensions from natural mineral molybdenite have been prepared in the emulsions microenvironment built by the water/surfactant/CO2 system. The size, thickness, and atomic structure are characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), and laser-scattering particle size analysis. Meanwhile, by the analysis of photoluminescence spectroscopy and microscope, the MoS2 nanosheets with smaller lateral dimensions exhibit extraordinary photoluminescence properties different from those with relatively larger lateral dimensions. The discovery of the excitation dependent photoluminescence for MoS2 nanosheets makes them potentially of interests for the applications in optoelectronics and biology. Moreover, we demonstrate that the fabricated MoS2 nanosheets can be a nontoxic fluorescent label for cell-targeted labeling application.

Open LED Illuminator: A Simple and Inexpensive LED Illuminator for Fast Multicolor Particle Tracking in Neurons

PubMed Central

Bosse, Jens B.; Tanneti, Nikhila S.; Hogue, Ian B.; Enquist, Lynn W.

2015-01-01

Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution. PMID:26600461

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2013-03-01

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

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

Single Molecule Spectroelectrochemistry of Interfacial Charge Transfer Dynamics In Hybrid Organic Solar Cell

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

Pan, Shanlin

2014-11-16

Our research under support of this DOE grant is focused on applied and fundamental aspects of model organic solar cell systems. Major accomplishments are: 1) we developed a spectroelectorchemistry technique of single molecule single nanoparticle method to study charge transfer between conjugated polymers and semiconductor at the single molecule level. The fluorescence of individual fluorescent polymers at semiconductor surfaces was shown to exhibit blinking behavior compared to molecules on glass substrates. Single molecule fluorescence excitation anisotropy measurements showed the conformation of the polymer molecules did not differ appreciably between glass and semiconductor substrates. The similarities in molecular conformation suggest thatmore » the observed differences in blinking activity are due to charge transfer between fluorescent polymer and semiconductor, which provides additional pathways between states of high and low fluorescence quantum efficiency. Similar spectroelectrochemistry work has been done for small organic dyes for understand their charge transfer dynamics on various substrates and electrochemical environments; 2) We developed a method of transferring semiconductor nanoparticles (NPs) and graphene oxide (GO) nanosheets into organic solvent for a potential electron acceptor in bulk heterojunction organic solar cells which employed polymer semiconductor as the electron donor. Electron transfer from the polymer semiconductor to semiconductor and GO in solutions and thin films was established through fluorescence spectroscopy and electroluminescence measurements. Solar cells containing these materials were constructed and evaluated using transient absorption spectroscopy and dynamic fluorescence techniques to understand the charge carrier generation and recombination events; 3) We invented a spectroelectorchemistry technique using light scattering and electroluminescence for rapid size determination and studying electrochemistry of single NPs in an electrochemical cell. For example, we are able to use this technique to track electroluminescence of single Au NPs, and the electrodeposition of individual Ag NPs in-situ. These metallic NPs are useful to enhance light harvesting in organic photovoltaic systems. The scattering at the surface of an indium tin oxide (ITO) working electrode was measured during a potential sweep. Utilizing Mie scattering theory and high resolution scanning electron microscopy (SEM), the scattering data were used to calculate current-potential curves depicting the electrodeposition of individual Ag NPs. The oxidation of individual presynthesized and electrodeposited Ag NPs was also investigated using fluorescence and DFS microscopies. Our work has produced 1 US provisional patent, 15 published manuscripts, 1 submitted and two additional in-writing manuscripts. 5 graduate students, 1 postdoctoral student, 1 visiting professor, and two undergraduate students have received research training in the area of electrochemistry and optical spectroscopy under support of this award.« less

Identification of distinct physiochemical properties of toxic prefibrillar species formed by A{beta} peptide variants

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

Goeransson, Anna-Lena, E-mail: anngo@ifm.liu.se; Nilsson, K. Peter R., E-mail: petni@ifm.liu.se; Kagedal, Katarina, E-mail: katarina.kagedal@liu.se

2012-04-20

Highlights: Black-Right-Pointing-Pointer Identification of toxic prefibrillar A{beta} species. Black-Right-Pointing-Pointer Fluorescence measurements using a combined set of fluorophores. Black-Right-Pointing-Pointer Morphology studies using transmission electron microscopy. -- Abstract: The formation of amyloid-{beta} peptide (A{beta}) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of A{beta}. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of A{beta}-derived peptides possessing different levels of neurotoxic activity,more » using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various A{beta} aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those A{beta} peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the A{beta} peptide to form nontoxic versus toxic species.« less

Spectroscopic studies on some fluorescent mixed-ligand titanium(IV) complexes.

PubMed

Baranwal, Balram Prasad; Singh, Alok Kumar; Varma, Anand

2011-12-15

A novel route to synthesize some titanium(IV) complexes containing acetylacetone, straight chain carboxylic acid and hydroxycarboxylic acid ligands has been investigated. Complexes with the general formula [Ti(acac)Cl(2-n)(OOCR*)(n)(OOCC(15)H(31))] (where Hacac=acetylacetone, R*COOH=hydroxycarboxylic acids and n=1 or 2) have been isolated and characterized. Molecular weight determinations indicated mononuclear nature of the complexes. LMCT bands were observed in the electronic spectra. Infrared spectra suggested bidentate nature of the ligands. Fluorescent behaviour of the complexes was noticed on the basis of fluorescence spectra. Powder XRD indicated them to be semi-crystalline having the crystallite size in 136-185 nm range. Transmission electron microscopy (TEM) indicated spherical particles of ~ 200 nm diameter. On the basis of physico-chemical studies, it is suggested that titanium is having coordination number 7 or 8 in these complexes. Copyright © 2011 Elsevier B.V. All rights reserved.

Engulfment of ceramic particles by fibroblasts does not alter cell behavior.

PubMed

Faye, Pierre-Antoine; Roualdes, Olivier; Rossignol, Fabrice; Hartmann, Daniel Jean; Desmoulière, Alexis

2017-02-17

Despite many studies, the impact of ceramic particles on cell behavior remains unclear. The aim of the present study was to investigate the effects of nano-sized ceramic particles on fibroblastic cells. Fibroblasts (dermal fibroblasts freshly isolated from skin samples and WI26 fibroblastic cells) were cultured in a monolayer in the presence of alumina or cerium-zirconia particles (≈50 nm diameter) at two concentrations (100 or 500 μg ml -1 ). Fluorescent alumina particles were also used. The following properties were analyzed: cell morphology, cytoplasmic ceramic incorporation (using confocal and transmission electron microscopy) and migration (using a silicon insert). Sedimentation field-flow fractionation (SdFFF) was also used to evaluate the rate of incorporation of ceramic particles into the cells. Finally, after treatment with various concentrations of ceramic particles, fibroblasts were also included in a collagen type I lattice constituting a dermal equivalent (DE), and the collagen lattice retraction and cell proliferation were evaluated. In monolayer conditions, the presence of both alumina and cerium-zirconia ceramic particles did not cause any deleterious effects on cultured cells (dermal fibroblast and WI26 cells) and cell fate was not affected in any way by the presence of ceramic particles in the cytoplasm. Confocal (using fluorescent alumina particles) and electron microscopy (using both alumina and cerium-zirconia particles) showed that ceramic particles were internalized in the WI26 cells. Using fluorescent membrane labeling and fluorescent alumina particles, a membrane was observed around the particle-containing vesicles present in the cytoplasm. Electron microscopy on WI26 cells showed the presence of a classical bilayer membrane around the ceramic particles. Interestingly, SdFFF confirmed that some dermal fibroblasts contained many alumina ceramic particles while others contained very few; in WI26 cells, the uptake of alumina ceramic was more homogeneous. In DE, collagen lattice retraction and cell proliferation were unchanged when WI26 fibroblastic cells contained alumina or cerium-zirconia ceramic particles. Our data suggest that ceramic particles are internalized in the cells by endocytosis. The presence of ceramic particles in the cytoplasm has no affect on cell behavior, confirming the excellent biocompatibility of this material and anticipating a minimal harmful effect of potential wear debris.

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

Determining the size of nanoparticles in the example of magnetic iron oxide core-shell systems

NASA Astrophysics Data System (ADS)

Jarzębski, Maciej; Kościński, Mikołaj; Białopiotrowicz, Tomasz

2017-08-01

The size of nanoparticles is one of the most important factors for their possible applications. Various techniques for the nanoparticle size characterization are available. In this paper selected techniques will be considered base on the prepared core-shell magnetite nanoparticles. Magnetite is one of the most investigated and developed magnetic material. It shows interesting magnetic properties which can be used for biomedical applications, such as drug delivery, hypothermia and also as a contrast agent. To reduce the toxic effects of Fe3O4, magnetic core was covered by dextran and gelatin. Moreover, the shell was doped by fluorescent dye for confocal microscopy investigation. The main investigation focused on the methods for particles size determination of modified magnetite nanoparticles prepared with different techniques. The size distribution were obtained by nanoparticle tracking analysis, dynamic light scattering and transmission electron microscopy. Furthermore, fluorescent correlation spectroscopy (FCS) and confocal microscopy were used to compare the results for particle size determination of core-shell systems.

Photothermal imaging of skeletal muscle mitochondria.

PubMed

Tomimatsu, Toru; Miyazaki, Jun; Kano, Yutaka; Kobayashi, Takayoshi

2017-06-01

The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers.

Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting

PubMed Central

Veatch, Sarah L.; Machta, Benjamin B.; Shelby, Sarah A.; Chiang, Ethan N.; Holowka, David A.; Baird, Barbara A.

2012-01-01

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins. PMID:22384026

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.

Filling carbon nanotubes with particles.

PubMed

Kim, Byong M; Qian, Shizhi; Bau, Haim H

2005-05-01

The filling of carbon nanotubes (CNTs) with fluorescent particles was studied experimentally and theoretically. The fluorescent signals emitted by the particles were visible through the walls of the nanotubes, and the particles inside the tubes were observable with an electron microscope. Taking advantage of the template-grown carbon nanotubes' transparency to fluorescent light, we measured the filling rate of the tubes with particles at room conditions. Liquids such as ethylene glycol, water, and ethylene glycol/water mixtures, laden with 50 nm diameter fluorescent particles, were brought into contact with 500 nm diameter CNTs. The liquid and the particles' transport were observed, respectively, with optical and fluorescence microscopy. The CNTs were filled controllably with particles by the complementary action of capillary forces and the evaporation of the liquid. The experimental results were compared and favorably agreed with theoretical predictions. This is the first report on fluorescence studies of particle transport in carbon nanotubes.

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

Atmospheric scanning electron microscope for correlative microscopy.

PubMed

Morrison, Ian E G; Dennison, Clare L; Nishiyama, Hidetoshi; Suga, Mitsuo; Sato, Chikara; Yarwood, Andrew; O'Toole, Peter J

2012-01-01

The JEOL ClairScope is the first truly correlative scanning electron and optical microscope. An inverted scanning electron microscope (SEM) column allows electron images of wet samples to be obtained in ambient conditions in a biological culture dish, via a silicon nitride film window in the base. A standard inverted optical microscope positioned above the dish holder can be used to take reflected light and epifluorescence images of the same sample, under atmospheric conditions that permit biochemical modifications. For SEM, the open dish allows successive staining operations to be performed without moving the holder. The standard optical color camera used for fluorescence imaging can be exchanged for a high-sensitivity monochrome camera to detect low-intensity fluorescence signals, and also cathodoluminescence emission from nanophosphor particles. If these particles are applied to the sample at a suitable density, they can greatly assist the task of perfecting the correlation between the optical and electron images. Copyright © 2012 Elsevier Inc. All rights reserved.

Chemical analyses of fossil bone.

PubMed

Zheng, Wenxia; Schweitzer, Mary Higby

2012-01-01

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

Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

PubMed Central

Ngo, John T.; Adams, Stephen R.; Deerinck, Thomas J.; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F.; Bertozzi, Carolyn R.; Ellisman, Mark H.; Tsien, Roger Y.

2016-01-01

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

Reversible Deformation of Transfusion Tracheids in Taxus baccata Is Associated with a Reversible Decrease in Leaf Hydraulic Conductance1[OPEN

PubMed Central

Zhang, Yong-Jiang; Rockwell, Fulton E.; Wheeler, James K.; Holbrook, N. Michele

2014-01-01

Declines in leaf hydraulic conductance (Kleaf) with increasing water stress have been attributed to cavitation of the leaf xylem. However, in the leaves of conifers, the reversible collapse of transfusion tracheids may provide an alternative explanation. Using Taxus baccata, a conifer species without resin, we developed a modified rehydration technique that allows the separation of declines in Kleaf into two components: one reversible and one irreversible upon relaxation of water potential to −1 MPa. We surveyed leaves at a range of water potentials for evidence of cavitation using cryo-scanning electron microscopy and quantified dehydration-induced structural changes in transfusion tracheids by cryo-fluorescence microscopy. Irreversible declines in Kleaf did not occur until leaf water potentials were more negative than −3 MPa. Declines in Kleaf between −2 and −3 MPa were reversible and accompanied by the collapse of transfusion tracheids, as evidenced by cryo-fluorescence microscopy. Based on cryo-scanning electron microscopy, cavitation of either transfusion or xylem tracheids did not contribute to declines in Kleaf in the reversible range. Moreover, the deformation of transfusion tracheids was quickly reversible, thus acting as a circuit breaker regulating the flux of water through the leaf vasculature. As transfusion tissue is present in all gymnosperms, the reversible collapse of transfusion tracheids may be a general mechanism in this group for the protection of leaf xylem from excessive loads generated in the living leaf tissue. PMID:24948828

Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice.

PubMed

Tang, Tao; Zhang, Lianfeng; Gao, Ran; Dai, Yunping; Meng, Fanchao; Li, Ying

2012-04-01

Bacterial magnetic particles (BMPs) are of interest as potential carriers of bioactive macromolecules, drugs, or liposomes. In this study, a high-pressure homogenizer was used to disrupt Magnetospirillum gryphiswaldense strain MSR-1 cells, and BMPs were purified. BMPs were labeled with fluorescence reagent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocianin perchlorate (DiI) and injected into the tail vein of BALB/c nude mice. Distribution of fluorescence signals of DiI-BMPs in vivo was examined using a whole-body fluorescence imaging system. The result showed that fluorescence signals were detected in liver, stomach, intestine, lungs, and spleen. However, transmission electron microscopy of ultrathin sections indicated that BMPs were mainly present in liver and lungs, but not in the other organs. BMPs could be useful as carriers for targeted drug therapy of diseases of the liver or lung.

The future of electron microscopy

DOE PAGES

Zhu, Yimei; Durr, Hermann

2015-04-01

Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifiesmore » to the importance of modern microscopy.« less

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

PubMed

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

2016-10-01

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

Studying aerosol light scattering based on aspect ratio distribution observed by fluorescence microscope.

PubMed

Li, Li; Zheng, Xu; Li, Zhengqiang; Li, Zhanhua; Dubovik, Oleg; Chen, Xingfeng; Wendisch, Manfred

2017-08-07

Particle shape is crucial to the properties of light scattered by atmospheric aerosol particles. A method of fluorescence microscopy direct observation was introduced to determine the aspect ratio distribution of aerosol particles. The result is comparable with that of the electron microscopic analysis. The measured aspect ratio distribution has been successfully applied in modeling light scattering and further in simulation of polarization measurements of the sun/sky radiometer. These efforts are expected to improve shape retrieval from skylight polarization by using directly measured aspect ratio distribution.

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

Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

PubMed

Huang, S K; Cheng, M; Hui, S W

1990-11-01

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.

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.

Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells.

PubMed

Kitchens, Kelly M; Foraker, Amy B; Kolhatkar, Rohit B; Swaan, Peter W; Ghandehari, Hamidreza

2007-11-01

To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.

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

Utility of fluorescence microscopy in embryonic/fetal topographical analysis.

PubMed

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

1995-06-01

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

Fluorescence properties of Yb3+-Er3+ co-doped phosphate glasses containing silver nanoparticles

NASA Astrophysics Data System (ADS)

Martínez Gámez, Ma A.; Vallejo H, Miguel A.; Kiryanov, A. V.; Licea-Jiménez, L.; Lucio M, J. L.; Pérez-García, S. A.

2018-04-01

Er3+-Yb3+ co-doped phosphate glasses containing silver nitrate (SN), were fabricated. Transmission electron microscopy (TEM) and x-ray photoelectron spectroscopy (XPS) analyses were used to evidence the nucleation and presence of silver nanoparticles (SNP). The basic parameters of the glasses were inspected by means of absorption and fluorescence spectra, and fluorescence lifetimes under excitation at 916 nm (in-band of Yb3+), and at 406 nm (in-band of surface plasmon resonance given by the presence of SNP). The spectra as well as estimates for the basic parameters defining the lasing/amplifying potential of the glasses were studied as a function of SN concentration. The experimental results indicate that by increasing the SN content an enhancement of Er3+/Yb3+ fluorescence takes place.

The preparation of ethylenediamine-modified fluorescent carbon dots and their use in imaging of cells.

PubMed

Dong, Wei; Zhou, Siqi; Dong, Yan; Wang, Jingwen; Ge, Xin; Sui, Lili

2015-09-01

In this work, fluorescent carbon dots (CDs) were synthesized using a hydrothermal method with glucose as the carbon source and were surface-modified with ethylenediamine. The properties of as-prepared CDs were analyzed by transmission electron microscopy (TEM), Fourier transform infrared (FTIR), ultraviolet-visible light (UV/vis) absorption and fluorescent spectra. Furthermore, CDs conjugated with mouse anti-(human carcinoembryonic antigen) (CEA) monoclonal antibody were successful employed in the biolabeling and fluorescent imaging of human gastric carcinoma cells. In addition, the cytotoxicity of CDs was also tested using human gastric carcinoma cells. There was no apparent cytotoxicity on human gastric carcinoma cells. These results suggest the potential application of the as-prepared CDs in bioimaging and related fields. Copyright © 2015 John Wiley & Sons, Ltd.

Viral capsid mobility: a dynamic conduit for inactivation.

PubMed

Broo, K; Wei, J; Marshall, D; Brown, F; Smith, T J; Johnson, J E; Schneemann, A; Siuzdak, G

2001-02-27

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

Microscopic Characterization of the Brazilian Giant Samba Virus.

PubMed

Schrad, Jason R; Young, Eric J; Abrahão, Jônatas S; Cortines, Juliana R; Parent, Kristin N

2017-02-14

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses .

Microscopic Characterization of the Brazilian Giant Samba Virus

PubMed Central

Schrad, Jason R.; Young, Eric J.; Abrahão, Jônatas S.; Cortines, Juliana R.; Parent, Kristin N.

2017-01-01

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses. PMID:28216551

Exopolysaccharide microchannels direct bacterial motility and organize multicellular behavior

DOE PAGES

Berleman, James E.; Zemla, Marcin; Remis, Jonathan P.; ...

2016-05-06

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required formore » cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS - mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus.« less

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.

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells.

PubMed

Wüstner, Daniel; Lund, Frederik W; Röhrl, Clemens; Stangl, Herbert

2016-01-01

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections.

PubMed

Moore, Chad L; Cheng, Delfine; Shami, Gerald J; Murphy, Christopher R

2016-05-01

In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Targeted Cancer Therapy: Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity (Adv. Healthcare Mater. 11/2016).

PubMed

Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

2016-06-01

On page 1310 J. S. Merzaban, A. E. Porter, and co-workers present fluorescently labeled RGD-targeted ZnO nanoparticles (NPs; green) for the targeted delivery of cytotoxic ZnO to integrin αvβ3 receptors expressed on triple negative breast cancer cells. Correlative light-electron microscopy shows that NPs dissolve into ionic Zn(2+) (blue) upon uptake and cause apoptosis (red) with intra-tumor heterogeneity, thereby providing a possible strategy for targeted breast cancer therapy. Cover design by Ivan Gromicho. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Size controlled biogenic silver nanoparticles as antibacterial agent against isolates from HIV infected patients

NASA Astrophysics Data System (ADS)

Suganya, K. S. Uma; Govindaraju, K.; Kumar, V. Ganesh; Dhas, T. Stalin; Karthick, V.; Singaravelu, G.; Elanchezhiyan, M.

2015-06-01

Silver nanoparticles (AgNPs) are synthesized using biological sources due to its high specificity in biomedical applications. Herein, we report the size and shape controlled synthesis of AgNPs using the aqueous extract of blue green alga, Spirulina platensis. Size, shape and elemental composition of AgNPs were characterized using UV-vis spectroscopy, Fluorescence spectroscopy, FT-IR (Fourier Transform-Infrared Spectroscopy), FT-RS (Fourier Transform-Raman Spectroscopy), SEM-EDAX (Scanning Electron Microscopy-Energy Dispersive X-ray analysis) and HR-TEM (High Resolution Transmission Electron Microscopy). AgNPs were stable, well defined and monodispersed (spherical) with an average size of 6 nm. The synthesized AgNPs were tested for its antibacterial potency against isolates obtained from HIV patients.

Designing of fluorescent and magnetic imprinted polymer for rapid, selective and sensitive detection of imidacloprid via activators regenerated by the electron transfer-atom transfer radical polymerization (ARGET-ATRP) technique

NASA Astrophysics Data System (ADS)

Kumar, Sunil; Karfa, Paramita; Madhuri, Rashmi; Sharma, Prashant K.

2018-05-01

In this work, we report on a dual-behavior electrochemical/optical sensor for sensitive determination of Imidacloprid by fluorescent dye (fluorescein, FL) and imprinted polymer modified europium doped superparamagnetic iron oxide nanoparticles (FL@SPIONs@MIP). The imidacloprid (IMD)-imprinted polymer was directly synthesized on the Eu-SPIONs surface via Activators regenerated by the electron transfer-atom transfer radical polymerization (ARGET-ATRP) technique. Preparation, characterization and application of the prepared FL@SPIONs@MIP were systematically investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), fluorescence spectroscopy and electrochemical techniques. The electrochemical experiments exhibited a remarkable selectivity of the prepared sensor towards IMD. Determination of IMD by the square wave stripping voltammetry method represented a wide linear range of 0.059-0.791 μg L-1 with a detection limit of 0.0125 μg L-1. In addition, the fluorescence method shows a linear range of 0.039-0.942 μg L-1 and LOD of 0.0108 μg L-1. The fluorescence property of prepared FL@SPIONs@MIP was used for rapid, on-spot but selective detection of IMD in real samples. The proposed electrode displayed excellent repeatability and long-term stability and was successfully applied for quantitative and trace level determination of IMD in several real samples.

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

PubMed Central

Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

2013-01-01

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology. PMID:23457228

Quick and simple estimation of bacteria using a fluorescent paracetamol dimer-Au nanoparticle composite

NASA Astrophysics Data System (ADS)

Sahoo, Amaresh Kumar; Sharma, Shilpa; Chattopadhyay, Arun; Ghosh, Siddhartha Sankar

2012-02-01

Rapid, simple and sensitive detection of bacterial contamination is critical for safeguarding public health and the environment. Herein, we report an easy method of detection as well as enumeration of the bacterial cell number on the basis of fluorescence quenching of a non-antibacterial fluorescent nanocomposite, consisting of paracetamol dimer (PD) and Au nanoparticles (NPs), in the presence of bacteria. The composite was synthesized by reaction of paracetamol (p-hydroxyacetanilide) with HAuCl4. The Au NPs of the composite were characterized using UV-Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction and selected area electron diffraction analysis. The paracetamol dimer in the composite showed emission peak at 435 nm when excited at 320 nm. The method successfully detected six bacterial strains with a sensitivity of 100 CFU mL-1. The Gram-positive and Gram-negative bacteria quenched the fluorescence of the composite differently, making it possible to distinguish between the two. The TEM analysis showed interaction of the composite with bacteria without any apparent damage to the bacteria. The chi-square test established the accuracy of the method. Quick, non-specific and highly sensitive detection of bacteria over a broad range of logarithmic dilutions within a short span of time demonstrates the potential of this method as an alternative to conventional methods.Rapid, simple and sensitive detection of bacterial contamination is critical for safeguarding public health and the environment. Herein, we report an easy method of detection as well as enumeration of the bacterial cell number on the basis of fluorescence quenching of a non-antibacterial fluorescent nanocomposite, consisting of paracetamol dimer (PD) and Au nanoparticles (NPs), in the presence of bacteria. The composite was synthesized by reaction of paracetamol (p-hydroxyacetanilide) with HAuCl4. The Au NPs of the composite were characterized using UV-Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction and selected area electron diffraction analysis. The paracetamol dimer in the composite showed emission peak at 435 nm when excited at 320 nm. The method successfully detected six bacterial strains with a sensitivity of 100 CFU mL-1. The Gram-positive and Gram-negative bacteria quenched the fluorescence of the composite differently, making it possible to distinguish between the two. The TEM analysis showed interaction of the composite with bacteria without any apparent damage to the bacteria. The chi-square test established the accuracy of the method. Quick, non-specific and highly sensitive detection of bacteria over a broad range of logarithmic dilutions within a short span of time demonstrates the potential of this method as an alternative to conventional methods. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11837h

The Application of Fluorescent Quantum Dots to Confocal, Multiphoton, and Electron Microscopic Imaging

PubMed Central

Deerinck, Thomas J.

2009-01-01

Fluorescent quantum dots are emerging as an important tool for imaging cells and tissues, and their unique optical and physical properties have captured the attention of the research community. The most common types of commercially available quantum dots consist of a nanocrystalline semiconductor core composed of cadmium selenide with a zinc sulfide capping layer and an outer polymer layer to facilitate conjugation to targeting biomolecules such as immunoglobulins. They exhibit high fluorescent quantum yields and have large absorption cross-sections, possess excellent photostability, and can be synthesized so that their narrow-band fluorescence emission can occur in a wide spectrum of colors. These properties make them excellent candidates for serving as multiplexing molecular beacons using a variety of imaging modalities including highly correlated microscopies. Whereas much attention has been focused on quantum-dot applications for live-cell imaging, we have sought to characterize and exploit their utility for enabling simultaneous multiprotein immunolabeling in fixed cells and tissues. Considerations for their application to immunolabeling for correlated light and electron microscopic analysis are discussed. PMID:18337229

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)☆

PubMed Central

Duke, Elizabeth M.H.; Razi, Minoo; Weston, Anne; Guttmann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Tooze, Sharon A.; Collinson, Lucy M.

2014-01-01

Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities. PMID:24238600

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.

A sensitive fluorescent nanosensor for chloramphenicol based on molecularly imprinted polymer-capped CdTe quantum dots.

PubMed

Amjadi, Mohammad; Jalili, Roghayeh; Manzoori, Jamshid L

2016-05-01

A novel fluorescent nanosensor using molecularly imprinted silica nanospheres embedded CdTe quantum dots (CdTe@SiO2 @MIP) was developed for detection and quantification of chloramphenicol (CAP). The imprinted sensor was prepared by synthesis of molecularly imprinting polymer (MIP) on the hydrophilic CdTe quantum dots via reverse microemulsion method using small amounts of solvents. The resulting CdTe@SiO2 @MIP nanoparticles were characterized by fluorescence, UV-vis absorption and FT-IR spectroscopy and transmission electron microscopy. They preserved 48% of fluorescence quantum yield of the parent quantum dots. CAP remarkably quenched the fluorescence of prepared CdTe@SiO2 @MIP, probably via electron transfer mechanism. Under the optimal conditions, the relative fluorescence intensity of CdTe@SiO2 @MIP decreased with increasing CAP by a Stern-Volmer type equation in the concentration range of 40-500 µg L(-1). The corresponding detection limit was 5.0 µg L(-1). The intra-day and inter-day values for the precision of the proposed method were all <4%. The developed sensor had a good selectivity and was applied to determine CAP in spiked human and bovine serum and milk samples with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.

Fluorescent Gold Nanoclusters for Selective Detection of Dopamine in Cerebrospinal fluid

PubMed Central

Govindaraju, Saravanan; Ankireddy, Seshadri Reddy; Viswanath, Buddolla; Kim, Jongsung; Yun, Kyusik

2017-01-01

Since the last two decades, protein conjugated fluorescent gold nanoclusters (NCs) owe much attention in the field of medical and nanobiotechnology due to their excellent photo stability characteristics. In this paper, we reported stable, nontoxic and red fluorescent emission BSA-Au NCs for selective detection of L-dopamine (DA) in cerebrospinal fluid (CSF). The evolution was probed by various instrumental techniques such as UV-vis spectroscopy, High resolution transmission electron microscopy (HTEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), photoluminescence spectroscopy (PL). The synthesised BSA-Au NCs were showing 4–6 nm with high fluorescent ~8% Quantum yield (QY). The fluorescence intensity of BSA-Au NCs was quenched upon the addition of various concentrations of DA via an electron transfer mechanism. The decrease in BSA-Au NCs fluorescence intensity made it possible to determine DA in PBS buffer and the spiked DA in CSF in the linear range from 0 to 10 nM with the limit of detection (LOD) 0.622 and 0.830 nM respectively. Best of our knowledge, as-prepared BSA-Au NCs will gain possible strategy and good platform for biosensor, drug discovery, and rapid disease diagnosis such as Parkinson’s and Alzheimer diseases. PMID:28067307

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

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.

Layer-by-layer films and colloidal dispersions of graphene oxide nanosheets for efficient control of the fluorescence and aggregation properties of the cationic dye acridine orange.

PubMed

Hansda, Chaitali; Chakraborty, Utsav; Hussain, Syed Arshad; Bhattacharjee, Debajyoti; Paul, Pabitra Kumar

2016-03-15

Chemically derived graphene oxide (GO) nanosheets have received great deal of interest for technological application such as optoelectronic and biosensors. Aqueous dispersions of GO become an efficient template to induce the association of cationic dye namely Acridine Orange (AO). Interactions of AO with colloidal GO was governed by both electrostatic and π-π stacking cooperative interactions. The type of dye aggregations was found to depend on the concentration of GO in the mixed ensemble. Spectroscopic calculations revealed the formation of both H and J-type dimers, but H-type aggregations were predominant. Preparation of layer-by-layer (LbL) electrostatic self-assembled films of AO and GO onto poly (allylamine hydrochloride) (PAH) coated quartz substrate is also reported in this article. UV-Vis absorption, steady state and time resolve fluorescence and Raman spectroscopic techniques have been employed to explore the detail photophysical properties of pure AO, AO/GO mixed solution and AO/GO LbL films. Scanning electron microscopy was also used for visual evidence of the synthesized nanodimensional GO sheets. The fluorescence quenching of AO in the presence of GO in aqueous solution was due to the interfacial photoinduced electron transfer (PET) from photoexcited AO to GO i.e. GO acts as an efficient quenching agent for the fluorescence emission of AO. The quenching is found to be static in nature. Raman spectroscopic results also confirmed the interaction of AO with GO and the electron transfer. The formation of AO/GO complex via very fast excited state electron transfer mechanism may be proposed as to prepare GO-based fluorescence sensor for biomolecular detection without direct labeling the biomolecules by fluorescent probe. Copyright © 2015 Elsevier B.V. All rights reserved.

Layer-by-layer films and colloidal dispersions of graphene oxide nanosheets for efficient control of the fluorescence and aggregation properties of the cationic dye acridine orange

NASA Astrophysics Data System (ADS)

Hansda, Chaitali; Chakraborty, Utsav; Hussain, Syed Arshad; Bhattacharjee, Debajyoti; Paul, Pabitra Kumar

2016-03-01

Chemically derived graphene oxide (GO) nanosheets have received great deal of interest for technological application such as optoelectronic and biosensors. Aqueous dispersions of GO become an efficient template to induce the association of cationic dye namely Acridine Orange (AO). Interactions of AO with colloidal GO was governed by both electrostatic and π-π stacking cooperative interactions. The type of dye aggregations was found to depend on the concentration of GO in the mixed ensemble. Spectroscopic calculations revealed the formation of both H and J-type dimers, but H-type aggregations were predominant. Preparation of layer-by-layer (LbL) electrostatic self-assembled films of AO and GO onto poly (allylamine hydrochloride) (PAH) coated quartz substrate is also reported in this article. UV-Vis absorption, steady state and time resolve fluorescence and Raman spectroscopic techniques have been employed to explore the detail photophysical properties of pure AO, AO/GO mixed solution and AO/GO LbL films. Scanning electron microscopy was also used for visual evidence of the synthesized nanodimensional GO sheets. The fluorescence quenching of AO in the presence of GO in aqueous solution was due to the interfacial photoinduced electron transfer (PET) from photoexcited AO to GO i.e. GO acts as an efficient quenching agent for the fluorescence emission of AO. The quenching is found to be static in nature. Raman spectroscopic results also confirmed the interaction of AO with GO and the electron transfer. The formation of AO/GO complex via very fast excited state electron transfer mechanism may be proposed as to prepare GO-based fluorescence sensor for biomolecular detection without direct labeling the biomolecules by fluorescent probe.

Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X-ray Fluorescence Imaging and Scanning Transmission X-ray Microscopy.

PubMed

Yang, Soo In; George, Graham N; Lawrence, John R; Kaminskyj, Susan G W; Dynes, James J; Lai, Barry; Pickering, Ingrid J

2016-10-04

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food web. Biofilms, naturally occurring microbial consortia, play numerous important roles in the environment, especially in biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally forming multispecies biofilms presents challenges for characterization because conventional microscopic techniques require chemical and physical modifications of the sample. Here, multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These complementary synchrotron techniques required minimal sample preparation and were applied correlatively to the same biofilm areas. Sub-micrometer XFI showed distributions of Se and endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of elemental Se (Se 0 ). Nanoscale carbon K-edge STXM revealed the distributions of microbial cells, extracellular polymeric substances (EPS), and lipids using the protein, saccharide, and lipid signatures, respectively, together with highly localized Se 0 using the Se L III edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50-700 nm, suggesting Se 0 nanoparticles. The intimate association of Se 0 particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment.

Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X-ray Fluorescence Imaging and Scanning Transmission X-ray Microscopy

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

Yang, Soo In; George, Graham N.; Lawrence, John R.

2016-10-04

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food web. Biofilms, naturally occurring microbial consortia, play numerous important roles in the environment, especially in biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally forming multispecies biofilms presents challenges for characterization because conventional microscopic techniques require chemical and physical modifications of the sample. Here, multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These complementary synchrotron techniques required minimal sample preparation and were applied correlatively to themore » same biofilm areas. Sub-micrometer XFI showed distributions of Se and endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of elemental Se (Se0). Nanoscale carbon K-edge STXM revealed the distributions of microbial cells, extracellular polymeric substances (EPS), and lipids using the protein, saccharide, and lipid signatures, respectively, together with highly localized Se0 using the Se LIII edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50–700 nm, suggesting Se0 nanoparticles. The intimate association of Se0 particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment.« less

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.

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

PubMed

Kijanka, M; van Donselaar, E G; Müller, W H; Dorresteijn, B; Popov-Čeleketić, D; El Khattabi, M; Verrips, C T; van Bergen En Henegouwen, P M P; Post, J A

2017-07-01

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM. Copyright © 2017. Published by Elsevier Inc.

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

Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling

NASA Astrophysics Data System (ADS)

Jahn, Martin T.; Markert, Sebastian M.; Ryu, Taewoo; Ravasi, Timothy; Stigloher, Christian; Hentschel, Ute; Moitinho-Silva, Lucas

2016-10-01

Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.

Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

PubMed Central

Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

2016-01-01

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy.

PubMed

Mainprize, Iain L; Beniac, Daniel R; Falkovskaia, Elena; Cleverley, Robert M; Gierasch, Lila M; Ottensmeyer, F Peter; Andrews, David W

2006-12-01

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.

Solvothermal preparation of phthalocyanine nanorod/rGO composites and their application to visible-light-responsive photocatalysts

NASA Astrophysics Data System (ADS)

Zhang, Shuai; Lu, Yongting; Zhang, Fan; Qu, Jie; Lin, Bencai; Yuan, Ningyi; Fang, Bijun; Ding, Jian-Ning

2016-09-01

Phthalocyanine (Pc) nanorod/reduced graphene oxide (rGO) composites were prepared by a simple solvothermal method, in which Pc nanosheet and graphene oxide (GO) suspensions were mixed in methanol. As characterized by scanning electron microscopy, transmission electron microscopy, and selected area electron diffraction, Pc nanorods with an amorphous structure and an average diameter of 250nm are partially covered by rGO sheets. In the photodegradation experiments, all the composites with different rGO content show enhanced photocatalytic activity for Rhodamine B decomposition under visible-light compared to pure Pc nanorods or rGO sheets. The enhanced photocatalytic activity shall be ascribed to the large surface area offered by rGO and the charge-transfer from Pc to rGO as indicated by the photoluminescence measurement, in which fluorescence intensity of the composites is much weaker than that of Pc nanorods.

Graphene Microcapsule Arrays for Combinatorial Electron Microscopy and Spectroscopy in Liquids

DOE PAGES

Yulaev, Alexander; Guo, Hongxuan; Strelcov, Evgheni; ...

2017-04-27

Atomic-scale thickness, molecular impermeability, low atomic number, and mechanical strength make graphene an ideal electron-transparent membrane for material characterization in liquids and gases with scanning electron microscopy and spectroscopy. Here in this paper, we present a novel sample platform made of an array of thousands of identical isolated graphene-capped microchannels with high aspect ratio. A combination of a global wide field of view with high resolution local imaging of the array allows for high throughput in situ studies as well as for combinatorial screening of solutions, liquid interfaces, and immersed samples. We demonstrate the capabilities of this platform by studyingmore » a pure water sample in comparison with alkali halide solutions, a model electrochemical plating process, and beam-induced crystal growth in liquid electrolyte. Spectroscopic characterization of liquid interfaces and immersed objects with Auger and X-ray fluorescence analysis through the graphene membrane are also demonstrated.« less

Development of multifunctional nanoparticles towards applications in non-invasive magnetic resonance imaging and axonal tracing.

PubMed

Du, Yan; Qin, Yubo; Li, Zizhen; Yang, Xiuying; Zhang, Jingchang; Westwick, Harrison; Tsai, Eve; Cao, Xudong

2017-12-01

A multifunctional nanobiomaterial has been developed by deliberately combining functions of superparamagnetism, fluorescence, and axonal tracing into one material. Superparamagnetic iron oxide nanoparticles were first synthesized and coated with a silica layer to prevent emission quenching through core-dye interactions; a fluorescent molecule, fluorescein isothiocyanate, was doped inside second layer of silica shell to improve photo-stability and to enable further thiol functionalization. Subsequently, biotinylated dextran amine, a sensitive axonal tracing reagent, was immobilized on the thiol-functionalized nanoparticle surfaces. The resulting nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, X-ray diffraction, X-ray photoelectron spectroscopy, UV-Vis spectroscopy, magnetic resonance imaging and fluorescence confocal microscopy. In vitro cell experiments using both undifferentiated and differentiated Neuro-2a cells showed that the cells were able to take up the nanoparticles intracellularly and that the nanoparticles showed good biocompatibilities. In summary, this new material demonstrated promising performances for both optical and magnetic resonance imaging modalities, suggesting its promising potentials in applications such as in non-invasive imaging, particularly in neuronal tracing.

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.

Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

NASA Astrophysics Data System (ADS)

Shinogle-Decker, Heather; Martinez-Rivera, Noraida; O'Brien, John; Powell, Richard D.; Joshi, Vishwas N.; Connell, Samuel; Rosa-Molinar, Eduardo

2018-02-01

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.

2-Hydroxy-naphthyl functionalized mesoporous silica for fluorescence sensing and removal of aluminum ions.

PubMed

Das, Trisha; Roy, Ankita; Uyama, Hiroshi; Roy, Partha; Nandi, Mahasweta

2017-06-06

Mesoporous silica functionalized with a 2-hydroxy-naphthyl moiety has been synthesized and characterized by standard techniques like powder X-ray diffraction, N 2 adsorption/desorption studies, transmission electron microscopy and spectral studies like FT-IR, UV-visible, fluorescence and 13 C and 29 Si solid state NMR. The functionalized silica material showed significant enhancement in its emission intensity in the presence of Al 3+ ions whereas other metal ions could not bring about any increase in its emission intensity. They either quench the emission or do not alter the intensity significantly making the functionalized material a fluorescence chemosensor for Al 3+ . The sensitivity of the probe towards Al 3+ has been determined to be high with a low limit of detection value. As functionalized silica is not soluble in common solvents, it has been effectively used to bind and remove Al 3+ from a solution. Theoretical calculations on a model system have been performed to investigate the electronic spectral transitions.

Evaluation of cell toxicity and DNA and protein binding of green synthesized silver nanoparticles.

PubMed

Ribeiro, A P C; Anbu, S; Alegria, E C B A; Fernandes, A R; Baptista, P V; Mendes, R; Matias, A S; Mendes, M; Guedes da Silva, M F C; Pombeiro, A J L

2018-05-01

Silver nanoparticles (AgNPs) were prepared by GREEN chemistry relying on the reduction of AgNO 3 by phytochemicals present in black tea extract. AgNPs were fully characterized by transmission electron microscopy (TEM), ultraviolet-visible spectroscopy ((UV-vis)), X-ray diffraction (XRD) and energy dispersive absorption spectroscopy (EDS). The synthesized AgNPs induced a decrease of the cell viability in a dose-dependent manner with a low IC 50 (0.5 ± 0.1 μM) for an ovarian carcinoma cell line (A2780) compared to primary human fibroblasts (IC 50 5.0 ± 0.1 μM). The DNA binding capability of CT (calf thymus) DNA was investigated using electronic absorption and fluorescence spectroscopies, circular dichroism and viscosity titration methods. Additionally, the AgNPs strongly quench the intrinsic fluorescence of BSA, as determined by synchronous fluorescence spectra. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Solid-phase synthesis of graphene quantum dots from the food additive citric acid under microwave irradiation and their use in live-cell imaging.

PubMed

Zhuang, Qianfen; Wang, Yong; Ni, Yongnian

2016-05-01

The work demonstrated that solid citric acid, one of the most common food additives, can be converted to graphene quantum dots (GQDs) under microwave heating. The as-prepared GQDs were further characterized by various analytical techniques like transmission electron microscopy, atomic force microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, fluorescence and UV-visible spectroscopy. Cytotoxicity of the GQDs was evaluated using HeLa cells. The result showed that the GQDs almost did not exhibit cytotoxicity at concentrations as high as 1000 µg mL(-1). In addition, it was found that the GQDs showed good solubility, excellent photostability, and excitation-dependent multicolor photoluminescence. Subsequently, the multicolor GQDs were successfully used as a fluorescence light-up probe for live-cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.

In Situ Observations of Electric-Field Induced Nanoparticle Aggregation

NASA Astrophysics Data System (ADS)

Woehl, T. J.; Browning, N. D.; Ristenpart, W. D.

2010-11-01

Nanoparticles have been widely observed to aggregate laterally on electrodes in response to applied electric fields. The mechanism driving this behavior, however, is unclear. Several groups have interpreted the aggregation in terms of electrohydrodynamic or electroosmotic fluid motion, but little corroborating evidence has been presented. Notably, work to date has relied on post situ observations using electron microscopy. Here we present a fluorescence microscopy technique to track the dynamics of nanoparticle aggregation in situ. Fluorescent 20-nm polystyrene nanoparticles are observed to form optically visible aggregates in response to an applied AC field. Although single particle resolution is lost, the existence of aggregates on the electrode surface is marked by growing clusters of increasingly bright intensity. We present a systematic investigation of the effects of applied potential and frequency on the aggregation rate, and we interpret the behavior in terms of a mechanism based on electrically induced convective flow.

Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

PubMed Central

Zhu, Xinyue; Wang, Jianxi; Zhang, Jianjian; Chen, Zhenjie; Zhang, Haixia; Zhang, Xiaoyu

2015-01-01

A reaction-based two-photon (TP) ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS) as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM). PMID:25594597

Synthesis and characterization of graphene quantum dots/cobalt ferrite nanocomposite

NASA Astrophysics Data System (ADS)

Ramachandran, Shilpa; Sathishkumar, M.; Kothurkar, Nikhil K.; Senthilkumar, R.

2018-02-01

A facile method has been developed for the synthesis of a graphene quantum dots/cobalt ferrite nanocomposite. Graphene quantum dots (GQDs) were synthesized by a simple bottom-up method using citric acid, followed by the co-precipitation of cobalt ferrite nanoparticles on the graphene quantum dots. The morphology, structural analysis, optical properties, magnetic properties were investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), UV-vis absorption spectroscopy, fluorescence spectroscopy, vibrating sample magnetometry (VSM) measurements. The synthesized nanocomposite showed good fluorescence and superparamagnetic properties, which are important for biomedical applications.

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.

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

NASA Astrophysics Data System (ADS)

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

2012-08-01

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

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.

Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin.

PubMed

Bhattacharya, Arpan; Bhowmik, Soumitra; Singh, Amit K; Kodgire, Prashant; Das, Apurba K; Mukherjee, Tushar Kanti

2017-10-10

The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-β-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of self-assembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.

Construction of dual-functional polymer nanomaterials with near-infrared fluorescence imaging and polymer prodrug by RAFT-mediated aqueous dispersion polymerization.

PubMed

Tian, Chun; Niu, Jinyun; Wei, Xuerui; Xu, Yujie; Zhang, Lifen; Cheng, Zhenping; Zhu, Xiulin

2018-05-31

The performance of functional polymer nanomaterials is a vigorously discussed topic in polymer science. We devoted ourselves to investigating polymer nanomaterials based on near-infrared (NIR) fluorescence imaging and polymer prodrug in this study. Aza-boron dipyrromethene (BODIPY) is an important organic dye, having characteristics such as environmental resistance, light resistance, high molar extinction coefficient, and fluorescence quantum yield. We incorporated it into our target monomer, which can be polymerized without changing its parent structure in a polar solvent and copolymerized with water-soluble monomer to improve the solubility of the dye in an aqueous solution. At the same time, the hydrophobic drug camptothecin (CPT) was designed as a prodrug monomer, and the polymeric nanoparticles (NPs) with NIR fluorescence imaging and prodrug were synthesized in situ in reversible addition-fragmentation chain transfer (RAFT)-mediated aqueous dispersion polymerization. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed the final uniform size of the dual-functional polymeric NPs morphology. The dual-functional polymeric NPs had a strong absorption and emission signal in the NIR region (>650 nm) based on the fluorescence tests. In consideration of the long-term biological toxicity, confocal laser scanning microscopy (CLSM) results indicated that the dual-functional NPs with controlled drug content exhibited effective capability of killing HeLa cells. In addition, in vivo imaging of the dual-functional NPs was observed in real time, and the fluorescent signals clearly demonstrated the dynamic process of prodrug transfer.

Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

NASA Astrophysics Data System (ADS)

Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

2017-10-01

Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

PubMed

Bharat, Tanmay A M; Hoffmann, Patrick C; Kukulski, Wanda

2018-04-10

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.

Characterization and Imaging of Antibody-Coated Gold Nanoparticles for Targeted Treatment of Microbial Keratitis

NASA Astrophysics Data System (ADS)

Mahan, Matthew

Microbial keratitis (MK) is an infection of the cornea by pathogenic organisms that causes inflammation and irritation. It can lead to full or partial blindness if left untreated. Current clinical treatment methods rely on high frequency application of topical drugs which are subject to the issues of patient compliance and microbial resistance. In this work, gold nanoparticles (AuNP) were proposed as an alternative treatment method in light-based therapies. Particle formulation methods were investigated and assessed using transmission electron microscopy (TEM) and ultraviolet/visible spectroscopy (UV-Vis). AuNP of 20 nm diameter were used as platforms to attach monoclonal antibodies anti-FLAG or anti-F1 to enhance their cell-targeting ability as well as polyethylene glycol to reduce non-specific binding and protein adsorption. These functionalized particles were qualitatively assessed using UV-Vis. The antibody-functionalized AuNP were then assessed for their ability to attach directly to Pseudomonas aeruginosa, expressing FLAG peptide, or Aspergillus fumigatus, expressing the F1 receptor. Attachment was imaged using dark field microscopy, transmission electron microscopy, and fluorescence microscopy.

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.

Green Synthesis of Novel Polyaniline Nanofibers: Application in pH Sensing.

PubMed

Tanwar, Shivani; Ho, Ja-an Annie

2015-10-13

An optically active polyaniline nanomaterial (PANI-Nap), doped with (S)-naproxen, was developed and evaluated as a potent pH sensor. We synthesized the material in one pot by the addition of the dopant, (S)-naproxen, prior to polymerization, followed by the addition of the oxidizing agent (ammonium persulfate) that causes polymerization of the aniline. This green chemistry approach allowed us to take only 1 h to produce a water-soluble and stable nanomaterial. UV-visible spectroscopy, fluorescence spectroscopy, FT-IR spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) were used to characterize the designed nanomaterial. This nanomaterial exhibited excellent pH sensing properties and showed long term stability (up to one month) without loss of sensor performance.

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

PubMed

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

2012-04-25

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

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.

Singlet oxygen production in Chlamydomonas reinhardtii under heat stress.

PubMed

Prasad, Ankush; Ferretti, Ursula; Sedlářová, Michaela; Pospíšil, Pavel

2016-02-01

In the current study, singlet oxygen formation by lipid peroxidation induced by heat stress (40 °C) was studied in vivo in unicellular green alga Chlamydomonas reinhardtii. Primary and secondary oxidation products of lipid peroxidation, hydroperoxide and malondialdehyde, were generated under heat stress as detected using swallow-tailed perylene derivative fluorescence monitored by confocal laser scanning microscopy and high performance liquid chromatography, respectively. Lipid peroxidation was initiated by enzymatic reaction as inhibition of lipoxygenase by catechol and caffeic acid prevented hydroperoxide formation. Ultra-weak photon emission showed formation of electronically excited species such as triplet excited carbonyl, which, upon transfer of excitation energy, leads to the formation of either singlet excited chlorophyll or singlet oxygen. Alternatively, singlet oxygen is formed by direct decomposition of hydroperoxide via Russell mechanisms. Formation of singlet oxygen was evidenced by the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl detected by electron paramagnetic resonance spin-trapping spectroscopy and the imaging of green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Suppression of singlet oxygen formation by lipoxygenase inhibitors indicates that singlet oxygen may be formed via enzymatic lipid peroxidation initiated by lipoxygenase.

Neuronal growth cones respond to laser-induced axonal damage

PubMed Central

Wu, Tao; Mohanty, Samarendra; Gomez-Godinez, Veronica; Shi, Linda Z.; Liaw, Lih-Huei; Miotke, Jill; Meyer, Ronald L.; Berns, Michael W.

2012-01-01

Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness (‘thinning’) within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model. PMID:21831892

Single-cell analysis of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy approach

NASA Astrophysics Data System (ADS)

LI, J.; Zhang, H.; Liu, P.; Menguy, N.; Pan, Y.

2017-12-01

Magnetotactic bacteria (MTB) are phylogenetically diverse and can biomineralize magnetic nanocrystals of magnetite or greigite in intracellular structures termed magnetosomes. Their remains within sediments or sedimentary rocks, i.e. magnetofossils, have been used to retrieve paleomagnetic and paleoenvironmental information of deposition time, as well as to trace the origin and evolution of life on Earth and even perhaps Mars. A precise identification of magnetofossils heavily depends on our knowledge of phylogenetic diversity and magnetosomal biomineralization within natural MTB. In this paper, we will present a novel method which can rapidly characterize both the phylogenetic and biomineralogical properties of uncultured MTB at the single-cell level by coupling fluorescence and electron microscopy. Using this method, we have successfully identified several uncultured MTB strains from natural environments in China. These MTB are phylogenetically affiliated with the Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria and Nitrospirae phylum, and form octahedral, cuboctahedral, prismatic, tooth-like and bullet-shaped magnetite magnetosomes. A corresponding analysis of magnetosome morphology and bacterial phylogenetics on each MTB strain has shown a species/strain-specific magnetosome biomineralization. The new method is not only promising for better understanding the correlation between magnetosome mineral habits and MTB phylogenies, but also crucial for unambiguously identifying magnetofossils.

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.

Lights Will Guide You : Sample Preparation and Applications for Integrated Laser and Electron Microscopy

NASA Astrophysics Data System (ADS)

Karreman, M. A.

2013-03-01

Correlative microscopy is the combined use of two different forms of microscopy in the study of a specimen, allowing for the exploitation of the advantages of both imaging tools. The integrated Laser and Electron Microscope (iLEM), developed at Utrecht University, combines a fluorescence microscope (FM) and a transmission electron microscope (TEM) in a single set-up. The region of interest in the specimen is labeled or tagged with a fluorescent probe and can easily be identified within a large field of view with the FM. Next, this same area is retraced in the TEM and can be studied at high resolution. The iLEM demands samples that can be imaged with both FM and TEM. Biological specimen, typically composed of light elements, generate low image contrast in the TEM. Therefore, these samples are often ‘contrasted’ with heavy metal stains. FM, on the other hand, images fluorescent samples. Sample preparation for correlative microscopy, and iLEM in particular, is complicated by the fact that the heavy metals stains employed for TEM quench the fluorescent signal of the probe that is imaged with FM. The first part of this thesis outlines preparation procedures for biological material yielding specimen that can be imaged with the iLEM. Here, approaches for the contrasting of thin sections of cells and tissue are introduced that do not affect the fluorescence signal of the probe that marks the region of interest. Furthermore, two novel procedures, VIS2FIXH and VIS2FIX­FS are described that allow for the chemical fixation of thin sections of cryo-immobilized material. These procedures greatly expedite the sample preparation process, and open up novel possibilities for the immuno-labeling of difficult antigens, eg. proteins and lipids that are challenging to preserve. The second part of this thesis describes applications of iLEM in research in the field of life and material science. The iLEM was employed in the study of UVC induced apoptosis (programmed cell death) of human umbilical vein endothelial cells. A novel, RNA containing body was identified in the nuclei of cells going through the various stages of the apoptotic process. Furthermore, we demonstrated the potential of iLEM in the study of Facio Scapulo Humeral Dystrophy (FSHD), the third most common form of inherited muscular dystrophy. In this study, diseased cells are identified based on the immuno-labeling of proteins associated with FSHD pathology. In the field of heterogeneous catalysis, a structural and functional characterization of Fluid Catalytic Cracking (FCC) particles was performed with iLEM. FCC particles are employed in petrochemical industry, where they catalyze the breakdown of large molecules in crude oil fractions into functional products with lower molecular weight, like gasoline. The catalytic sites in the FCC particles were selectively stained with a fluorescent probe, and next their structure was investigated with TEM. The iLEM allowed for the identification and characterization of catalytically active areas in the FCC particles. Furthermore, a unique study of the deactivation processes taking place in an industrial FCC unit was performed by analyzing a sample derived from a FCC reactor

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

Contribution of high-resolution correlative imaging techniques in the study of the liver sieve in three-dimensions.

PubMed

Braet, Filip; Wisse, Eddie; Bomans, Paul; Frederik, Peter; Geerts, Willie; Koster, Abraham; Soon, Lilian; Ringer, Simon

2007-03-01

Correlative microscopy has become increasingly important for the analysis of the structure, function, and dynamics of cells. This is largely due to the result of recent advances in light-, probe-, laser- and various electron microscopy techniques that facilitate three-dimensional studies. Furthermore, the improved understanding in the past decade of imaging cell compartments in the third dimension has resulted largely from the availability of powerful computers, fast high-resolution CCD cameras, specifically developed imaging analysis software, and various probes designed for labeling living and or fixed cells. In this paper, we review different correlative high-resolution imaging methodologies and how these microscopy techniques facilitated the accumulation of new insights in the morpho-functional and structural organization of the hepatic sieve. Various aspects of hepatic endothelial fenestrae regarding their structure, origin, dynamics, and formation will be explored throughout this paper by comparing the results of confocal laser scanning-, correlative fluorescence and scanning electron-, atomic force-, and whole-mount electron microscopy. Furthermore, the recent advances of vitrifying cells with the vitrobot in combination with the glove box for the preparation of cells for cryo-electron microscopic investigation will be discussed. Finally, the first transmission electron tomography data of the liver sieve in three-dimensions are presented. The obtained data unambiguously show the involvement of special domains in the de novo formation and disappearance of hepatic fenestrae, and focuses future research into the (supra)molecular structure of the fenestrae-forming center, defenestration center and fenestrae-, and sieve plate cytoskeleton ring by using advanced cryo-electron tomography. (c) 2007 Wiley-Liss, Inc.

Effect of time discretization of the imaging process on the accuracy of trajectory estimation in fluorescence microscopy

PubMed Central

Wong, Yau; Chao, Jerry; Lin, Zhiping; Ober, Raimund J.

2014-01-01

In fluorescence microscopy, high-speed imaging is often necessary for the proper visualization and analysis of fast subcellular dynamics. Here, we examine how the speed of image acquisition affects the accuracy with which parameters such as the starting position and speed of a microscopic non-stationary fluorescent object can be estimated from the resulting image sequence. Specifically, we use a Fisher information-based performance bound to investigate the detector-dependent effect of frame rate on the accuracy of parameter estimation. We demonstrate that when a charge-coupled device detector is used, the estimation accuracy deteriorates as the frame rate increases beyond a point where the detector’s readout noise begins to overwhelm the low number of photons detected in each frame. In contrast, we show that when an electron-multiplying charge-coupled device (EMCCD) detector is used, the estimation accuracy improves with increasing frame rate. In fact, at high frame rates where the low number of photons detected in each frame renders the fluorescent object difficult to detect visually, imaging with an EMCCD detector represents a natural implementation of the Ultrahigh Accuracy Imaging Modality, and enables estimation with an accuracy approaching that which is attainable only when a hypothetical noiseless detector is used. PMID:25321248

Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.

PubMed

Rubino, L; Di Franco, A; Russo, M

2000-01-01

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

PubMed

Michalet, X; Colyer, R A; Scalia, G; Ingargiola, A; Lin, R; Millaud, J E; Weiss, S; Siegmund, Oswald H W; Tremsin, Anton S; Vallerga, John V; Cheng, A; Levi, M; Aharoni, D; Arisaka, K; Villa, F; Guerrieri, F; Panzeri, F; Rech, I; Gulinatti, A; Zappa, F; Ghioni, M; Cova, S

2013-02-05

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Development of new photon-counting detectors for single-molecule fluorescence microscopy

PubMed Central

Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

2013-01-01

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

Development of magnetic luminescent core/shell nanocomplex particles with fluorescence using Rhodamine 6G

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

Lee, Hee Uk; Song, Yoon Seok; Park, Chulhwan

2012-12-15

Graphical abstract: Display Omitted Highlights: ► A simple method was developed to synthesize Co-B/SiO{sub 2}/dye/SiO{sub 2} composite particles. ► The magnetic particle shows that highly luminescent and core/shell particles are formed. ► Such core/shell particles can be easily suspended in water. ► The magnetic particles could detect fluorescence for the application of biosensor. -- Abstract: A simple and reproducible method was developed to synthesize a novel class of Co-B/SiO{sub 2}/dye/SiO{sub 2} composite core/shell particles. Using a single cobalt core, Rhodamine 6G of organic dye molecules was entrapped in a silica shell, resulting in core/shell particles of ∼200 nm diameter. Analysesmore » using a variety of techniques such as transmission electron microscopy, X-ray photoelectron spectroscopy, vibration sample magnetometry, confocal laser scanning microscopy, and fluorescence intensity demonstrated that dye molecules were trapped inside the core/shell particles. A photoluminescence investigation showed that highly luminescent and photostable core/shell particles were formed. Such core/shell particles can be easily suspended in water. The synthesized magnetic particles could be used to detect fluorescence on glass substrate arrays for bioassay and biosensor applications.« less

Secondary sulfate minerals from Alum Cave Bluff: Microscopy and microanalysis

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

Lauf, R.J.

1997-07-01

Microcrystals of secondary sulfate minerals from Alum Cave Bluff, Great Smoky Mountains National Park, were examined by scanning electron microscopy and identified by X-ray fluorescence (XRF) in the SEM. Among the samples the author discovered three new rare-earth sulfates: coskrenite-(Ce), levinsonite-(Y), and zugshunstite-(Ce). Other minerals illustrated in this report include sulfur, tschermigite, gypsum, epsomite, melanterite, halotrichite, apjohnite, jarosite, slavikite, magnesiocopiapite, and diadochite. Additional specimens whose identification is more tentative include pickeringite, aluminite, basaluminite, and botryogen. Alum Cave is a ``Dana locality`` for apjohnite and potash alum, and is the first documented North American occurrence of slavikite.

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

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.

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

PubMed Central

Herfst, Sander; Bestebroer, Theo M.; Vaes, Vincent P.; van der Hoeven, Barbara; Koster, Abraham J.; Kremers, Gert-Jan; Scott, Dana P.; Gultyaev, Alexander P.; Sorell, Erin M.; de Graaf, Miranda; Bárcena, Montserrat; Rimmelzwaan, Guus F.; Fouchier, Ron A.

2015-01-01

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. PMID:26241861

Lichens survive in space: results from the 2005 LICHENS experiment.

PubMed

Sancho, Leopoldo G; de la Torre, Rosa; Horneck, Gerda; Ascaso, Carmen; de Los Rios, Asunción; Pintado, Ana; Wierzchos, J; Schuster, M

2007-06-01

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

Lichens Survive in Space: Results from the 2005 LICHENS Experiment

NASA Astrophysics Data System (ADS)

Sancho, Leopoldo G.; de la Torre, Rosa; Horneck, Gerda; Ascaso, Carmen; de los Rios, Asunción; Pintado, Ana; Wierzchos, J.; Schuster, M.

2007-06-01

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

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.

Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

PubMed Central

Kenworthy, A.K.; Edidin, M.

1998-01-01

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins. PMID:9660864

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.

A New Low Cost Wide-Field Illumination Method for Photooxidation of Intracellular Fluorescent Markers

PubMed Central

da Silva Filho, Manoel; Santos, Daniel Valle Vasconcelos; Costa, Kauê Machado

2013-01-01

Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our “photooxidizer” consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy. PMID:23441199

Nanospheres Encapsulating Anti-Leishmanial Drugs for Their Specific Macrophage Targeting, Reduced Toxicity, and Deliberate Intracellular Release

PubMed Central

Shukla, Anil Kumar; Patra, Sanjukta

2012-01-01

Abstract The current work focuses on the study of polymeric, biodegradable nanoparticles (NPs) for the encapsulation of doxorubicin and mitomycin C (anti-leishmanial drugs), and their efficient delivery to macrophages, the parasite's home. The biodegradable polymer methoxypoly-(ethylene glycol)-b-poly (lactic acid) (MPEG-PLA) was used to prepare polymeric NPs encapsulating doxorubicin and mitomycin C. The morphology, mean diameter, and surface area of spherical NPs were determined by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), and BET surface area analysis. X-ray diffraction was performed to validate drug encapsulation. An in vitro release profile of the drugs suggested a fairly slow release. These polymeric NPs were efficiently capable of releasing drug inside macrophages at a slower pace than the free drug, which was monitored by epi-fluorescence microscopy. Encapsulation of doxorubicin and mitomycin C into NPs also decreases cellular toxicity in mouse macrophages (J774.1A). PMID:22925019

A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface.

PubMed

Buser, Dominik P; Schleicher, Kai D; Prescianotto-Baschong, Cristina; Spiess, Martin

2018-06-18

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans -Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters' homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans -Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

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.

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

PubMed Central

Vielreicher, M.; Schürmann, S.; Detsch, R.; Schmidt, M. A.; Buttgereit, A.; Boccaccini, A.; Friedrich, O.

2013-01-01

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging. PMID:23864499

Synthesis and characterization of RuS2 nanostructures.

PubMed

Díaz, David; Castillo-Blum, Silvia E; Alvarez-Fregoso, Octavio; Rodríguez-Gattorno, Geonel; Santiago-Jacinto, Patricia; Rendon, Luis; Ortiz-Frade, Luis; León-Paredes, Yolia-Judith

2005-12-08

Small naked ruthenium sulfide nanoparticles (NPs) with narrow size distribution (2.5 +/- 0.4 nm of diameter) were synthesized in DMSO colloidal dispersions, under mild reaction conditions and using commercial RuCl3 as precursor. To test the chemical reactivity with soft and hard bases, fresh presynthesized RuS2 colloids were mixed with triethylamine (N(Et)3) and ammonium tetrathiomolybdate ((NH4)2MoS4) dimethyl sulfoxide solutions. Naked N(Et)3 and [MoS4](2-)-capped RuS2 nanoparticle colloids were characterized using UV-visible electronic absorption and emission spectroscopies and high-resolution transmission electron microscopy (HR-TEM). It has also been shown that capped RuS2-[MoS4]2- nanoparticles yield MoO3 crystalline matrix by means of HR-TEM experiments. The emission spectra of RuS2 and N(Et)3-RuS2 dispersions show that both nanosized materials have strong fluorescence. The existence of the ruthenium precursor species in solution was established by cyclic voltammetry. Moreover, naked RuS2 NPs were mixed with a chemical mixture with composition similar to gasoline (dibenzothiophene (Bz2S, 400 ppm), hexane, and toluene (55:45% v/v)). The reaction mixture consisted of two phases; in the polar phase, we found evidences of a strong interaction of Bz2S and toluene with the naked RuS2 NPs. We have also obtained self-organized thin films of capped N(Et)3- and RuS2-[MoS4]2- nanoparticles. In both cases, the shape and thickness of the resulting thin films were controlled by a dynamic vacuum procedure. The thin films have been characterized by atomic force microscopy, scanning electron microscopy, HR-TEM, energy dispersion spectroscopy, X-ray diffraction, and absorbance and fluorescence spectroscopies.

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

PubMed

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

2017-06-14

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

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.

Patterning nanoparticles into rings by "2-D Pickering emulsions".

PubMed

Lee, Cheol Hee; Crosby, Alfred J; Hayward, Ryan C; Emrick, Todd

2014-04-09

We present a simple method for the two-dimensional self-assembly of CdSe/ZnS quantum dots (QDs) into well-defined rings at the air/water interface, through the formation of "2-D Pickering emulsions". Surfactant molecules assemble at the air/water interface into islands that are subsequently surrounded by adsorption of QDs from the aqueous subphase. The QD rings emanating from this process range from ∼100 nm to several micrometers in diameter, as characterized by atomic force microscopy, scanning electron microscopy, and fluorescence microscopy. The deposition and alignment of QD rings onto large areas (cm(2)) were demonstrated by dip-coating onto a substrate. This simple method produces rings of QDs without the need for any templating or fabrication steps.

Assembly and microscopic characterization of DNA origami structures.

PubMed

Scheible, Max; Jungmann, Ralf; Simmel, Friedrich C

2012-01-01

DNA origami is a revolutionary method for the assembly of molecular nanostructures from DNA with precisely defined dimensions and with an unprecedented yield. This can be utilized to arrange nanoscale components such as proteins or nanoparticles into pre-defined patterns. For applications it will now be of interest to arrange such components into functional complexes and study their geometry-dependent interactions. While commonly DNA nanostructures are characterized by atomic force microscopy or electron microscopy, these techniques often lack the time-resolution to study dynamic processes. It is therefore of considerable interest to also apply fluorescence microscopic techniques to DNA nanostructures. Of particular importance here is the utilization of novel super-resolved microscopy methods that enable imaging beyond the classical diffraction limit.

Connecting quantum dots and bionanoparticles in hybrid nanoscale ultra-thin films

NASA Astrophysics Data System (ADS)

Tangirala, Ravisubhash; Hu, Yunxia; Zhang, Qingling; He, Jinbo; Russell, Thomas; Emrick, Todd

2008-03-01

Aldehyde-functionalized CdSe quantum dots and nanorods, and horse spleen ferritin bionanoparticles, were co-assembled at an oil-water interface. Reaction of the aldehydes with the surface-available amines on the ferritin particles enabled cross-linking at the interface, converting the assembled nanoparticles into robust ultra-thin films. The cross-linked capsules and sheets thus made by aldehyde-amine conjugation could be disrupted by addition of acid. Reductive amination chemistry could be performed to convert these degradable capsules and sheets into structures with irreversible cross-linking. Fluorescence confocal microscopy, scanning force microscopy and pendant drop tensiometry were used to characterize these hybrid nanoparticle-based materials, and transmission electron microscopy (TEM) confirmed the presence of both the synthetic and naturally derived nanoparticles.

A dual-stimuli-responsive fluorescent switch ultrathin film

NASA Astrophysics Data System (ADS)

Li, Zhixiong; Liang, Ruizheng; Liu, Wendi; Yan, Dongpeng; Wei, Min

2015-10-01

Stimuli-responsive fluorescent switches have shown broad applications in optical devices, biological materials and intelligent responses. Herein, we describe the design and fabrication of a dual-stimuli-responsive fluorescent switch ultrathin film (UTF) via a three-step layer-by-layer (LBL) technique: (i) encapsulation of spiropyran (SP) within an amphiphilic block copolymer (PTBEM) to give the (SP@PTBEM) micelle; (ii) the mixture of riboflavin (Rf) and poly(styrene 4-sulfonate) (PSS) to enhance the adhesion ability of small molecules; (iii) assembly of negatively charged SP@PTBEM and Rf-PSS with cationic layered double hydroxide (LDH) nanoplatelets to obtain the (Rf-PSS/LDH/SP@PTBEM)n UTFs (n: bilayer number). The assembly process of the UTFs and their luminescence properties, as monitored by fluorescence spectroscopy and scanning electron microscopy (SEM), present a uniform and ordered layered structure with stepwise growth. The resulting Rf-PSS/LDH/SP@PTBEM UTF serves as a three-state switchable multicolor (green, yellow, and red) luminescent system based on stimulation from UV/Vis light and pH, with an acceptable reversibility. Therefore, this work provides a facile way to fabricate stimuli-responsive solid-state film switches with tunable-color luminescence, which have potential applications in the areas of displays, sensors, and rewritable optical memory and fluorescent logic devices.Stimuli-responsive fluorescent switches have shown broad applications in optical devices, biological materials and intelligent responses. Herein, we describe the design and fabrication of a dual-stimuli-responsive fluorescent switch ultrathin film (UTF) via a three-step layer-by-layer (LBL) technique: (i) encapsulation of spiropyran (SP) within an amphiphilic block copolymer (PTBEM) to give the (SP@PTBEM) micelle; (ii) the mixture of riboflavin (Rf) and poly(styrene 4-sulfonate) (PSS) to enhance the adhesion ability of small molecules; (iii) assembly of negatively charged SP@PTBEM and Rf-PSS with cationic layered double hydroxide (LDH) nanoplatelets to obtain the (Rf-PSS/LDH/SP@PTBEM)n UTFs (n: bilayer number). The assembly process of the UTFs and their luminescence properties, as monitored by fluorescence spectroscopy and scanning electron microscopy (SEM), present a uniform and ordered layered structure with stepwise growth. The resulting Rf-PSS/LDH/SP@PTBEM UTF serves as a three-state switchable multicolor (green, yellow, and red) luminescent system based on stimulation from UV/Vis light and pH, with an acceptable reversibility. Therefore, this work provides a facile way to fabricate stimuli-responsive solid-state film switches with tunable-color luminescence, which have potential applications in the areas of displays, sensors, and rewritable optical memory and fluorescent logic devices. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05376e

Tryptophan and ATTO 590: mutual fluorescence quenching and exciplex formation.

PubMed

Bhattacharjee, Ujjal; Beck, Christie; Winter, Arthur; Wells, Carson; Petrich, Jacob W

2014-07-24

Investigation of fluorescence quenching of probes, such as ATTO dyes, is becoming an increasingly important topic owing to the use of these dyes in super-resolution microscopies and in single-molecule studies. Photoinduced electron transfer is their most important nonradiative pathway. Because of the increasing frequency of the use of ATTO and related dyes to investigate biological systems, studies are presented for inter- and intramolecular quenching of ATTO 590 with tryptophan. In order to examine intramolecular quenching, an ATTO 590-tryptophan conjugate was synthesized. It was determined that tryptophan is efficiently quenching ATTO 590 fluorescence by excited-state charge transfer and two charge transfer complexes are forming. In addition, it was discovered that an exciplex (whose lifetime is 5.6 ns) can be formed between tryptophan and ATTO 590, and it is suggested that the possibility of such exciplex formation should be taken into account when protein fluorescence is monitored in a system tagged with ATTO dyes.

Fluorescence Determination of Warfarin Using TGA-capped CdTe Quantum Dots in Human Plasma Samples.

PubMed

Dehbozorgi, A; Tashkhourian, J; Zare, S

2015-11-01

In this study, some effort has been performed to provide low temperature, less time consuming and facile routes for the synthesis of CdTe quantum dots using ultrasound and water soluble capping agent thioglycolic acid. TGA-capped CdTe quantum dots were characterized through x-ray diffraction, transmission electron microscopy, Fourier transform infrared, ultraviolet-visible and fluorescence spectroscopy. The prepared quantum dots were used for warfarin determination based on the quenching of the fluorescence intensity in aqueous solution. Under the optimized conditions, the linear range of quantum dots fluorescence intensity versus the concentration of warfarin was 0.1-160.0 μM, with the correlation coefficient of 0.9996 and a limit of detection of 77.5 nM. There was no interference to coexisting foreign substances. The selectivity of the sensor was also tested and the results show that the developed method possesses a high selectivity for warfarin.

Make Caffeine Visible: a Fluorescent Caffeine “Traffic Light” Detector

NASA Astrophysics Data System (ADS)

Xu, Wang; Kim, Tae-Hyeong; Zhai, Duanting; Er, Jun Cheng; Zhang, Liyun; Kale, Anup Atul; Agrawalla, Bikram Keshari; Cho, Yoon-Kyoung; Chang, Young-Tae

2013-07-01

Caffeine has attracted abundant attention due to its extensive existence in beverages and medicines. However, to detect it sensitively and conveniently remains a challenge, especially in resource-limited regions. Here we report a novel aqueous phase fluorescent caffeine sensor named Caffeine Orange which exhibits 250-fold fluorescence enhancement upon caffeine activation and high selectivity. Nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy indicate that π-stacking and hydrogen-bonding contribute to their interactions while dynamic light scattering and transmission electron microscopy experiments demonstrate the change of Caffeine Orange ambient environment induces its fluorescence emission. To utilize this probe in real life, we developed a non-toxic caffeine detection kit and tested it for caffeine quantification in various beverages. Naked-eye sensing of various caffeine concentrations was possible based on color changes upon irradiation with a laser pointer. Lastly, we performed the whole system on a microfluidic device to make caffeine detection quick, sensitive and automated.

Synthesis of positively charged CdTe quantum dots and detection for uric acid

NASA Astrophysics Data System (ADS)

Zhang, Tiliang; Sun, Xiangying; Liu, Bin

2011-09-01

The CdTe dots (QDs) coated with 2-Mercaptoethylamine was prepared in aqueous solution and characterized with fluorescence spectroscopy, UV-Vis absorption spectra, high-resolution transmission electron microscopy and infrared spectroscopy. When the λex = 350 nm, the fluorescence peak of positively charged CdTe quantum dots is at 592 nm. The uric acid is able to quench their fluorescence. Under optimum conditions, the change of fluorescence intensity is linearly proportional to the concentration of uric acid in the range 0.4000-3.600 μmol L -1, and the limit of detection calculated according to IUPAC definitions is 0.1030 μmol L -1. Compared with routine method, the present method determines uric acid in human serum with satisfactory results. The mechanism of this strategy is due to the interaction of the tautomeric keto/hydroxyl group of uric acid and the amino group coated at the CdTe QDs.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

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

Pham, Son; CSIRO Australian Animal Health Laboratory, Victoria 3220; Tabarin, Thibault

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstratemore » that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.« less

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…

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

PubMed Central

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

2011-01-01

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

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.

Diverse Protocols for Correlative Super-Resolution Fluorescence Imaging and Electron Microscopy of Cells and Tissue

DTIC Science & Technology

2016-05-25

tissue is critical to biology. Many factors determine optimal experimental design, including attainable localization precision, ultrastructural...both imaging modalities. Examples include: weak tissue preservation protocols resulting in poor ultrastructure, e.g. mitochondrial cristae membranes...tension effects during sample drying that may result in artifacts44. Samples dried in the presence of polyvinyl alcohol do not have the haziness

Application of several physical techniques in the total analysis of a canine urinary calculus.

PubMed

Rodgers, A L; Mezzabotta, M; Mulder, K J; Nassimbeni, L R

1981-06-01

A single calculus from the bladder of a Beagle bitch has been analyzed by a multiple technique approach employing x-ray diffraction, infrared spectroscopy, scanning electron microscopy, x-ray fluorescence spectrometry, atomic absorption spectrophotometry and density gradient fractionation. The qualitative and quantitative data obtained showed excellent agreement, lending confidence to such an approach for the evaluation and understanding of stone disease.

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

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

NASA Astrophysics Data System (ADS)

Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

2016-12-01

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

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.

Inexpensive electronics and software for photon statistics and correlation spectroscopy.

PubMed

Gamari, Benjamin D; Zhang, Dianwen; Buckman, Richard E; Milas, Peker; Denker, John S; Chen, Hui; Li, Hongmin; Goldner, Lori S

2014-07-01

Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors.

Inexpensive electronics and software for photon statistics and correlation spectroscopy

PubMed Central

Gamari, Benjamin D.; Zhang, Dianwen; Buckman, Richard E.; Milas, Peker; Denker, John S.; Chen, Hui; Li, Hongmin; Goldner, Lori S.

2016-01-01

Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors. PMID:26924846

Nanostructural origin of blue fluorescence in the mineral karpatite.

PubMed

Potticary, Jason; Jensen, Torsten T; Hall, Simon R

2017-08-29

The colour of crystals is a function of their atomic structure. In the case of organic crystals, it is the spatial relationships between molecules that determine the colour, so the same molecules in the same arrangement should produce crystals of the same colour, regardless of whether they arise geologically or synthetically. There is a naturally-occurring organic crystal known as karpatite which is prized for its beautiful blue fluorescence under ultra-violet illumination. When grown under laboratory conditions however, the crystals fluoresce with an intense green colour. For 20 years, this difference has been thought to be due to chemical impurities in the laboratory-grown material. Using electron microscopy coupled with fluorescence spectroscopy and X-Ray diffraction, we report here that this disparity is instead due to differences in the structure of the crystals at the nanoscale. The results show that in nature, karpatite has a nanotexture that is not present in the synthetic crystals, which enables different photonic pathways and therefore a blue, rather than green colour whilst undergoing fluorescence.

One-step synthesis of solid state luminescent carbon-based silica nanohybrids for imaging of latent fingerprints

NASA Astrophysics Data System (ADS)

Li, Feng; Li, Hongren; Cui, Tianfang

2017-11-01

Fluorescent carbon-based nanomaterials(CNs) with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. Despite the successes in preparing strongly fluorescent CNs, preserving the luminescence in solid materials is still challenging because of the serious emission quenching of CNs in solid state materials. In this work, fluorescent carbon and silica nanohybrids (SiCNHs) were synthesized via a simple one-step hydrothermal approach by carbonizing sodium citrate and (3-aminopropyl)triethoxysilane(APTES), and hydrolysis of tetraethyl orthosilicate(TEOS). The resultant SiCNs were characterized through X-ray diffraction (XRD), transmission electron microscopy (TEM), FT-IR, X-ray photoelectron spectroscopy (XPS), and photoluminescence (PL) spectroscopy. The SiCNs exhibited strong fluorescence in both aqueous and solid states. The luminescent solid state SiCNs power were successfully used as a fluorescent labeling material for enhanced imaging of latent fingerprints(LFPs) on single background colour and multi-coloured surfaces substrates in forensic science for individual identification.

Colour and surface fluorescence development and their relationship with Maillard reaction markers as influenced by structural changes during cornflakes production.

PubMed

Farroni, Abel; Buera, María Del Pilar

2012-12-01

The aim of this work was to study colour and surface fluorescence development in relation to the chemical markers for the Maillard reaction at the cooking, flaking and toasting stages of cornflake production process. Colour was measured by a calibrated computer vision system. Surface fluorescence was measured on compressed samples. Aqueous extracted Maillard reaction markers (hydroxymethylfurfural, carboxymethyl-lysine, absorbance at 420nm and total fluorescence) were measured on protease hydrolyzed samples. Sample microstructure was observed by scanning electron microscopy. During cooking the colour coordinates L(∗) and b(∗) decreased and a(∗) increased. After flaking, the samples appeared lighter, while the pigment concentration, fluorescence and hydroxymethylfurfural did not change. Toasting generated bubbles in the matrix and L(∗) apparently increased, although brown pigment concentration increased. Pigment concentration did not correlate with surface colour due to the destruction or generation of interfaces. Surface and microstructure effects can be avoided by milling and compressing the samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

Hybrid photodetector for single-molecule spectroscopy and microscopy

PubMed Central

Michalet, X.; Cheng, Adrian; Antelman, Joshua; Suyama, Motohiro; Arisaka, Katsushi; Weiss, Shimon

2011-01-01

We report benchmark tests of a new single-photon counting detector based on a GaAsP photocathode and an electron-bombarded avalanche photodiode developed by Hamamatsu Photonics. We compare its performance with those of standard Geiger-mode avalanche photodiodes. We show its advantages for FCS due to the absence of after-pulsing and for fluorescence lifetime measurements due to its excellent time resolution. Its large sensitive area also greatly simplifies setup alignment. Its spectral sensitivity being similar to that of recently introduced CMOS SPADs, this new detector could become a valuable tool for single-molecule fluorescence measurements, as well as for many other applications. PMID:21822361

Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

PubMed

Kraus, Max-Joseph; Seifert, Jan; Strasser, Erwin F; Gawaz, Meinrad; Schäffer, Tilman E; Rheinlaender, Johannes

2016-09-01

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading.

Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy and Live Cell Imaging

PubMed Central

Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Wasteneys, Geoffrey O.

2016-01-01

Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

Synthesis, physicochemical and optical properties of bis-thiosemicarbazone functionalized graphene oxide

NASA Astrophysics Data System (ADS)

Kumar, Santosh; Wani, Mohmmad Y.; Arranja, Claudia T.; Castro, Ricardo A. E.; Paixão, José A.; Sobral, Abilio J. F. N.

2018-01-01

Fluorescent materials are important for low-cost opto-electronic and biomedical sensor devices. In this study we present the synthesis and characterization of graphene modified with bis-thiosemicarbazone (BTS). This new material was characterized using Fourier transform infrared spectroscopy (FT-IR), Ultraviolet-visible (UV-Vis) and Raman spectroscopy techniques. Further evaluation by X-ray diffraction (XRD), thermo-gravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and atomic-force microscopy (AFM) allowed us to fully characterize the morphology of the fabricated material. The average height of the BTSGO sheet is around 10 nm. Optical properties of BTSGO evaluated by photoluminescence (PL) spectroscopy showed red shift at different excitation wavelength compared to graphene oxide or bisthiosemicarbazide alone. These results strongly suggest that BTSGO material could find potential applications in graphene based optoelectronic devices.

Leaching behavior and ESEM characterization of water-sensitive mudstone in southwestern Taiwan.

PubMed

Chen, Hung-Ta; Lin, Tzong-Tzeng; Chang, Juu-En

2003-05-01

This investigation attempts to understand the critical soluble salts in natural mudstone and the leaching, microstructural, and microchemical characteristics in soaked mudstone using scanning electron microscopy (SEM)/energy-dispersive X-ray analysis (EDAX), X-ray fluorescence spectrometry (XRF), X-ray diffractometry (XRD), conductivity measurement, ion chromatography (IC), and environmental scanning electron microscopy (ESEM)/EDAX techniques. Natural mudstone probably includes soluble salts such as Na2SO4, NaCl, NaCO3, and CaCO3. The dissolution of Na2SO4 controls water-sensitive mudstone very susceptible to slaking and dispersion. ESEM micrographs clearly show evidence of mudstone-slaking during soaking since the visible pores are filled with small aggregative masses. A calcium-bearing precipitate from the soaked mudstone is speculated to be attributable to the decomposition of the hydrated product of the fresh mudstone.

Fluorescence of fungi in superficial and deep fungal infections

PubMed Central

Elston, Dirk M

2001-01-01

Background Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique. Results In most cases, fluorescence was noted, but was not intense. Fluorescence of fungi did not correlate with the age of the specimen. In most cases, organisms in H&E stained sections were more easily identified with routine light microscopy than with fluorescent microscopy. Conclusion This report suggests that in H&E stained skin specimens, fluorescent microscopy is of little benefit in the identification of fungal organisms. PMID:11602016

Green biosynthesis of silver nanoparticles using Curcuma longa tuber powder

PubMed Central

Shameli, Kamyar; Ahmad, Mansor Bin; Zamanian, Ali; Sangpour, Parvanh; Shabanzadeh, Parvaneh; Abdollahi, Yadollah; Zargar, Mohsen

2012-01-01

Green synthesis of noble metal nanoparticles is a vastly developing area of research. Metallic nanoparticles have received great attention from chemists, physicists, biologists, and engineers who wish to use them for the development of a new-generation of nanodevices. In this study, silver nanoparticles were biosynthesized from aqueous silver nitrate through a simple and eco-friendly route using Curcuma longa tuber-powder extracts, which acted as a reductant and stabilizer simultaneously. Characterizations of nanoparticles were done using different methods, which included ultraviolet-visible spectroscopy, powder X-ray diffraction, transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray fluorescence spectrometry, and Fourier-transform infrared spectroscopy. The ultraviolet-visible spectrum of the aqueous medium containing silver nanoparticles showed an absorption peak at around 415 nm. Transmission electron microscopy showed that mean diameter and standard deviation for the formation of silver nanoparticles was 6.30 ± 2.64 nm. Powder X-ray diffraction showed that the particles are crystalline in nature, with a face-centered cubic structure. The most needed outcome of this work will be the development of value-added products from C. longa for biomedical and nanotechnology-based industries. PMID:23341739

Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

PubMed Central

Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

2014-01-01

Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid) with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM), two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery. PMID:24872922

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.

Electronically tunable femtosecond all-fiber optical parametric oscillator for multi-photon microscopy

NASA Astrophysics Data System (ADS)

Hellwig, Tim; Brinkmann, Maximilian; Fallnich, Carsten

2018-02-01

We present a femtosecond fiber-based optical parametric oscillator (FOPO) for multiphoton microscopy with wavelength tuning by electronic repetition rate tuning in combination with a dispersive filter in the FOPO cavity. The all-spliced, all-fiber FOPO cavity is based on polarization-maintaining fibers and a broadband output coupler, allowing to get access to the resonant signal pulses as well as the idler pulses simultaneously. The system was pumped by a gain-switched fiber-coupled laser diode emitting pulses at a central wavelength of 1030 nm and an electronically tunable repetition frequency of about 2 MHz. The pump pulses were amplified in an Ytterbium fiber amplifier system with a pulse duration after amplification of 13 ps. Tuning of the idler (1140 nm - 1300 nm) and signal wavelengths (850 nm - 940 nm) was achieved by changing the repetition frequency of the pump laser by about 4 kHz. The generated signal pulses reached a pulse energy of up to 9.2 nJ at 920 nm and were spectrally broadened to about 6 nm in the FOPO by a combination of self-phase and cross-phase modulation. We showed external compression of the idler pulses at 920 nm to about 430 fs and appleid them to two-photon excitation microscopy with green fluorescent dyes. The presented system constitutes an important step towards a fully fiber-integrated all-electronically tunable and, thereby, programmable light source and already embodies a versatile and flexible light source for applications, e.g., for smart microscopy.

The MIND PALACE: A Multi-Spectral Imaging and Spectroscopy Database for Planetary Science

NASA Astrophysics Data System (ADS)

Eshelman, E.; Doloboff, I.; Hara, E. K.; Uckert, K.; Sapers, H. M.; Abbey, W.; Beegle, L. W.; Bhartia, R.

2017-12-01

The Multi-Instrument Database (MIND) is the web-based home to a well-characterized set of analytical data collected by a suite of deep-UV fluorescence/Raman instruments built at the Jet Propulsion Laboratory (JPL). Samples derive from a growing body of planetary surface analogs, mineral and microbial standards, meteorites, spacecraft materials, and other astrobiologically relevant materials. In addition to deep-UV spectroscopy, datasets stored in MIND are obtained from a variety of analytical techniques obtained over multiple spatial and spectral scales including electron microscopy, optical microscopy, infrared spectroscopy, X-ray fluorescence, and direct fluorescence imaging. Multivariate statistical analysis techniques, primarily Principal Component Analysis (PCA), are used to guide interpretation of these large multi-analytical spectral datasets. Spatial co-referencing of integrated spectral/visual maps is performed using QGIS (geographic information system software). Georeferencing techniques transform individual instrument data maps into a layered co-registered data cube for analysis across spectral and spatial scales. The body of data in MIND is intended to serve as a permanent, reliable, and expanding database of deep-UV spectroscopy datasets generated by this unique suite of JPL-based instruments on samples of broad planetary science interest.

Localization and Visualization of a Coxiella-Type Symbiont within the Lone Star Tick, Amblyomma americanum▿ †

PubMed Central

Klyachko, Olga; Stein, Barry D.; Grindle, Nathan; Clay, Keith; Fuqua, Clay

2007-01-01

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens. PMID:17720830

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

PubMed

Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

2018-02-01

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Synthesis and Characterization of Reduced Graphene Oxide/Rhodamine 101 (rGO-Rh101) Nanocomposites and Their Heterojunction Performance in rGO-Rh101/ p-Si Device Configuration

NASA Astrophysics Data System (ADS)

Batır, G. Güven; Arık, Mustafa; Caldıran, Zakir; Turut, Abdulmecit; Aydogan, Sakir

2018-01-01

Reduced graphene oxide (rGO)-rhodamine 101 (Rh101) nanocomposites with different ratios of rGO have been synthesized in aqueous medium by ultrasonic homogenization. The fluorescence of Rh101 as measured using a laser dye with high fluorescence quantum yield was substantially quenched with increasing amount of rGO in the nanocomposite. Formation of rGO-Rh101 nanocomposites was confirmed by x-ray diffraction analysis, scanning electron microscopy, ultraviolet-visible (UV-Vis) spectroscopy, and fluorescence microscopy. Furthermore, rGO-Rh101 nanocomposite/ p-Si heterojunctions were synthesized, all of which showed good rectifying behavior. The electrical characteristics of these devices were analyzed using current-voltage ( I- V) measurements to determine the ideality factor and barrier height. The experimental results confirmed the presence of lateral inhomogeneity in the effective barrier height of the rGO-Rh101 nanocomposite/ p-Si heterojunctions. In addition to I- V measurements, one device was analyzed in more detail using frequency-dependent capacitance-voltage measurements. All electrical measurements were carried out at room temperature and in the dark.

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope

PubMed Central

Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

2015-01-01

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy. PMID:26819828

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope.

PubMed

Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

2016-01-01

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

PubMed

Fuchs, Felix M; Raguse, Marina; Fiebrandt, Marcel; Madela, Kazimierz; Awakowicz, Peter; Laue, Michael; Stapelmann, Katharina; Moeller, Ralf

2017-11-30

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Facile synthesis of Curcuma longa tuber powder engineered metal nanoparticles for bioimaging applications

NASA Astrophysics Data System (ADS)

Sankar, Renu; Rahman, Pattanathu K. S. M.; Varunkumar, Krishnamoorthy; Anusha, Chidambaram; Kalaiarasi, Arunachalam; Shivashangari, Kanchi Subramanian; Ravikumar, Vilwanathan

2017-02-01

Nanomaterials based fluorescent agents are rapidly becoming significant and promising transformative tools for improving medical diagnostics for extensive in vivo imaging modalities. Compared with conventional fluorescent agents, nano-fluorescence has capabilities to improve the in vivo detection and enriched targeting efficiencies. In our laboratory we synthesized fluorescent metal nanoparticles of silver, copper and iron using Curcuma longa tuber powder by simple reduction. The physicochemical properties of the synthesized metal nanoparticles were attained using UV-visible spectrophotometry, scanning electron microscopy with EDAX spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy and X-ray diffraction. The Curcuma longa tuber powder has one of the bioactive compound Curcumin might act as a capping agent during the synthesis of nanoparticles. The synthesized metal nanoparticles fluorescence property was confirmed by spectrofluorometry. When compared with copper and iron nanoparticles the silver nanoparticles showed high fluorescence intensity under spectrofluorometry. Moreover, in vitro cell images of the silver nanoparticles in A549 cell lines also correlated with the results of spectrofluorometry. These silver nanoparticles show inspiring cell-imaging applications. They enter into cells without any further modifications, and the fluorescence property can be utilized for fluorescence-based cell imaging applications.

Anionic poly(p-phenylenevinylene)/layered double hydroxide ordered ultrathin films with multiple quantum well structure: a combined experimental and theoretical study.

PubMed

Yan, Dongpeng; Lu, Jun; Ma, Jing; Wei, Min; Wang, Xinrui; Evans, David G; Duan, Xue

2010-05-18

The sulfonated phenylenevinylene polyanion derivate (APPV) and exfoliated Mg-Al-layered double hydroxide (LDH) monolayers were alternatively assembled into ordered ultrathin films (UTFs) employing a layer-by-layer method, which shows uniform yellow luminescence. UV-vis absorption and fluorescence spectroscopy present a stepwise and regular growth of the UTFs upon increasing deposited cycles. X-ray diffraction, atomic force microscopy, and scanning electron microscopy demonstrate that the UTFs are orderly periodical layered structure with a thickness of 3.3-3.5 nm per bilayer. The APPV/LDH UTFs exhibit well-defined polarized photoemission characteristic with the maximum luminescence anisotropy of approximately 0.3. Moreover, the UTF exhibit longer fluorescence lifetime (3-3.85-fold) and higher photostability than the drop-casting APPV film under UV irradiation, suggesting that the existence of a LDH monolayer enhances the optical performance of the APPV polyanion. A combination study of electrochemistry and periodic density functional theory was used to investigate the electronic structure of the APPV/LDH system, illustrating that the APPV/LDH UTF is a kind of organic-inorganic hybrid multiple quantum well (MQW) structure with a low band energy of 1.7-1.8 eV, where the valence electrons of APPV can be confined into the energy wells formed by the LDH monolayers effectively. Therefore, this work not only gives a feasible method for fabricating a luminescence ultrathin film but also provides a detailed understanding of the geometric and electronic structures of photoactive polyanions confined between the LDH monolayers.

Electronic cameras for low-light microscopy.

PubMed

Rasnik, Ivan; French, Todd; Jacobson, Ken; Berland, Keith

2013-01-01

This chapter introduces to electronic cameras, discusses the various parameters considered for evaluating their performance, and describes some of the key features of different camera formats. The chapter also presents the basic understanding of functioning of the electronic cameras and how these properties can be exploited to optimize image quality under low-light conditions. Although there are many types of cameras available for microscopy, the most reliable type is the charge-coupled device (CCD) camera, which remains preferred for high-performance systems. If time resolution and frame rate are of no concern, slow-scan CCDs certainly offer the best available performance, both in terms of the signal-to-noise ratio and their spatial resolution. Slow-scan cameras are thus the first choice for experiments using fixed specimens such as measurements using immune fluorescence and fluorescence in situ hybridization. However, if video rate imaging is required, one need not evaluate slow-scan CCD cameras. A very basic video CCD may suffice if samples are heavily labeled or are not perturbed by high intensity illumination. When video rate imaging is required for very dim specimens, the electron multiplying CCD camera is probably the most appropriate at this technological stage. Intensified CCDs provide a unique tool for applications in which high-speed gating is required. The variable integration time video cameras are very attractive options if one needs to acquire images at video rate acquisition, as well as with longer integration times for less bright samples. This flexibility can facilitate many diverse applications with highly varied light levels. Copyright © 2007 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.

Grain wall boundaries in centimeter-scale continuous monolayer WS2 film grown by chemical vapor deposition.

PubMed

Jia, Zhiyan; Hu, Wentao; Xiang, Jianyong; Wen, Fusheng; Nie, Anmin; Mu, Congpu; Zhao, Zhisheng; Xu, Bo; Tian, Yongjun; Liu, Zhongyuan

2018-06-22

Centimeter-scale continuous monolayer WS 2 film with large tensile strain has been successfully grown on oxidized silicon substrate by chemical vapor deposition, in which monolayer grains can be more than 200 μm in size. Monolayer WS 2 grains are observed to merge together via not only traditional grain boundaries but also non-traditional ones, which are named as grain walls (GWs) due to their nanometer-scale widths. The GWs are revealed to consist of two or three layers. Though not a monolayer, the GWs exhibit significantly enhanced fluorescence and photoluminescence. This enhancement may be attributed to abundant structural defects such as stacking faults and partial dislocations in the GWs, which are clearly observable in atomically resolved high resolution transmission electron microscopy and scanning transmission electron microscopy images. Moreover, GW-based phototransistor is found to deliver higher photocurrent than that based on monolayer film. These features of GWs provide a clue to microstructure engineering of monolayer WS 2 for specific applications in (opto)electronics.

Grain wall boundaries in centimeter-scale continuous monolayer WS2 film grown by chemical vapor deposition

NASA Astrophysics Data System (ADS)

Jia, Zhiyan; Hu, Wentao; Xiang, Jianyong; Wen, Fusheng; Nie, Anmin; Mu, Congpu; Zhao, Zhisheng; Xu, Bo; Tian, Yongjun; Liu, Zhongyuan

2018-06-01

Centimeter-scale continuous monolayer WS2 film with large tensile strain has been successfully grown on oxidized silicon substrate by chemical vapor deposition, in which monolayer grains can be more than 200 μm in size. Monolayer WS2 grains are observed to merge together via not only traditional grain boundaries but also non-traditional ones, which are named as grain walls (GWs) due to their nanometer-scale widths. The GWs are revealed to consist of two or three layers. Though not a monolayer, the GWs exhibit significantly enhanced fluorescence and photoluminescence. This enhancement may be attributed to abundant structural defects such as stacking faults and partial dislocations in the GWs, which are clearly observable in atomically resolved high resolution transmission electron microscopy and scanning transmission electron microscopy images. Moreover, GW-based phototransistor is found to deliver higher photocurrent than that based on monolayer film. These features of GWs provide a clue to microstructure engineering of monolayer WS2 for specific applications in (opto)electronics.

Effect of replacing Sn4+ ions by Zn2+ ions on structural, optical and magnetic properties of SnO2 nanoparticles

NASA Astrophysics Data System (ADS)

Selvi, E. Thamarai; Sundar, S. Meenakshi

2017-05-01

This paper highlights on the consequence of replacing tetravalent Sn4+ ions of the SnO2 by divalent Zn2+ ions on their structural, optical, and magnetic properties. Samples of Sn1- x Zn x O2 with x = 0, 0.01, 0.02, 0.03, and 0.04 were synthesized using microwave irradiated solvothermal process. The X-ray powder diffraction patterns reveal the rutile tetragonal phase of all doped SnO2 samples with no secondary phases. The transmission electron microscopy results show the formation of spherical nanoparticles of size 10-30 nm. Morphological changes were observed by scanning electron microscopy. The functional groups were investigated using Fourier transform infrared spectroscopy studies. Optical studies were carried by UV-Vis spectroscopy and fluorescence spectroscopy. Electron paramagnetic resonance was used to calculate the Lande splitting factor ` g'. The magnetic properties using vibrating sample magnetometer exhibit room temperature ferromagnetism for all the samples.

Imaging electronic trap states in perovskite thin films with combined fluorescence and femtosecond transient absorption microscopy

DOE PAGES

Xiao, Kai; Ma, Ying -Zhong; Simpson, Mary Jane; ...

2016-04-22

Charge carrier trapping degrades the performance of organometallic halide perovskite solar cells. To characterize the locations of electronic trap states in a heterogeneous photoactive layer, a spatially resolved approach is essential. Here, we report a comparative study on methylammonium lead tri-iodide perovskite thin films subject to different thermal annealing times using a combined photoluminescence (PL) and femtosecond transient absorption microscopy (TAM) approach to spatially map trap states. This approach coregisters the initially populated electronic excited states with the regions that recombine radiatively. Although the TAM images are relatively homogeneous for both samples, the corresponding PL images are highly structured. Themore » remarkable variation in the PL intensities as compared to transient absorption signal amplitude suggests spatially dependent PL quantum efficiency, indicative of trapping events. Furthermore, detailed analysis enables identification of two trapping regimes: a densely packed trapping region and a sparse trapping area that appear as unique spatial features in scaled PL maps.« less

High-speed particle tracking in microscopy using SPAD image sensors

NASA Astrophysics Data System (ADS)

Gyongy, Istvan; Davies, Amy; Miguelez Crespo, Allende; Green, Andrew; Dutton, Neale A. W.; Duncan, Rory R.; Rickman, Colin; Henderson, Robert K.; Dalgarno, Paul A.

2018-02-01

Single photon avalanche diodes (SPADs) are used in a wide range of applications, from fluorescence lifetime imaging microscopy (FLIM) to time-of-flight (ToF) 3D imaging. SPAD arrays are becoming increasingly established, combining the unique properties of SPADs with widefield camera configurations. Traditionally, the photosensitive area (fill factor) of SPAD arrays has been limited by the in-pixel digital electronics. However, recent designs have demonstrated that by replacing the complex digital pixel logic with simple binary pixels and external frame summation, the fill factor can be increased considerably. A significant advantage of such binary SPAD arrays is the high frame rates offered by the sensors (>100kFPS), which opens up new possibilities for capturing ultra-fast temporal dynamics in, for example, life science cellular imaging. In this work we consider the use of novel binary SPAD arrays in high-speed particle tracking in microscopy. We demonstrate the tracking of fluorescent microspheres undergoing Brownian motion, and in intra-cellular vesicle dynamics, at high frame rates. We thereby show how binary SPAD arrays can offer an important advance in live cell imaging in such fields as intercellular communication, cell trafficking and cell signaling.

Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin.

PubMed

Nostro, Antonia; Scaffaro, Roberto; Ginestra, Giovanna; D'Arrigo, Manuela; Botta, Luigi; Marino, Andreana; Bisignano, Giuseppe

2010-06-01

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Effect of size on structural, optical and magnetic properties of SnO2 nanoparticles

NASA Astrophysics Data System (ADS)

Thamarai Selvi, E.; Meenakshi Sundar, S.

2017-07-01

Tin Oxide (SnO2) nanostructures were synthesized by a microwave oven assisted solvothermal method using with and without cetyl trimethyl ammonium bromide (CTAB) capping agent. XRD confirmed the pure rutile-type tetragonal phase of SnO2 for both uncapped and capped samples. The presence of functional groups was analyzed by Fourier transform infrared spectroscopy. Scanning electron microscopy shows the morphology of the samples. Transmission electron microscopy images exposed the size of the SnO2 nanostructures. Surface defect-related g factor of SnO2 nanoparticles using fluorescence spectroscopy is shown. For both uncapped and capped samples, UV-visible spectrum shows a blue shift in absorption edge due to the quantum confinement effect. Defect-related bands were identified by electron paramagnetic resonance (EPR) spectroscopy. The magnetic properties were studied by using vibrating sample magnetometer (VSM). A high value of magnetic moment 0.023 emu g-1 at room temperature for uncapped SnO2 nanoparticles was observed. Capping with CTAB enhanced the saturation magnetic moment to high value of 0.081 emu g-1 by altering the electronic configuration on the surface.

Folate-grafted boron nitride nanotubes: possible exploitation in cancer therapy.

PubMed

Ferreira, Tiago H; Marino, Attilio; Rocca, Antonella; Liakos, Ioannis; Nitti, Simone; Athanassiou, Athanassia; Mattoli, Virgilio; Mazzolai, Barbara; de Sousa, Edesia M B; Ciofani, Gianni

2015-03-15

Boron nitride nanotubes (BNNTs) have generated considerable interest among the scientific community because of their unique physical and chemical properties. They present good chemical inertness, high thermal stability, and optimal resistance to oxidation, that make them ideal candidates for biomedical applications, in particular as nanovectors for drug, gene and protein delivery into the cells. In this study, BNNTs were prepared through a synthesis based on a chemical vapor deposition (CVD) method, and thereafter chemically functionalized with folic acid. The obtained nanostructures have been characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The characterization showed efficiently functionalized BNNTs of length of about 1 μm. Furthermore, confocal laser microscopy demonstrated that our nanotubes can be fluorescently-traced under appropriate excitation. Thanks to this property, it has been possible to investigate their internalization by HeLa cells through confocal microscopy, demonstrating that the BNNT up-take clearly increases after the functionalization with folate, a result confirmed by inductively coupled plasma (ICP) assessment of boron content inside the treated cell cultures. Copyright © 2015 Elsevier B.V. All rights reserved.

Form follows function: ultrastructure of different morphotypes of Physarum polycephalum

NASA Astrophysics Data System (ADS)

Oettmeier, Christina; Lee, Jonghyun; Döbereiner, Hans-Günther

2018-04-01

The multinucleate, unicellular slime mold Physarum polycephalum is a highly motile and morphologically diverse giant amoeba. Despite being brainless and lacking neurons, it exhibits ‘smart’ behavior. There is considerable interest in describing such traits and to investigate the underlying mechanochemical patterns which may hint at universal principles of behavior and decision-making. Furthermore, the slime mold’s mechanism of locomotion is unique. It resembles amoeboid movement, but differs from the locomotion of other amoebae in many ways, e.g. in their much larger size and lack of lobopodia. These two aspects, behavior and locomotion, are linked by the cytoskeleton and the overall morphology of P. polycephalum. In this paper, we present a structural analysis of different growth forms (micro-, meso- and macroplasmodia) by transmission electron microscopy (TEM), scanning electron microscopy (SEM), light microscopy, and fluorescence microscopy of F-actin. With these detailed investigations of cellular ultrastructure and morphology, we provide the basis for the analysis of, e.g. viscoelastic and rheological measurements. Our data also provide structural details for the many models that have been constructed for the understanding of locomotion. We conclude that morphological information is vital for the assessment and measurement of material properties.

Mixedness determination of rare earth-doped ceramics

NASA Astrophysics Data System (ADS)

Czerepinski, Jennifer H.

The lack of chemical uniformity in a powder mixture, such as clustering of a minor component, can lead to deterioration of materials properties. A method to determine powder mixture quality is to correlate the chemical homogeneity of a multi-component mixture with its particle size distribution and mixing method. This is applicable to rare earth-doped ceramics, which require at least 1-2 nm dopant ion spacing to optimize optical properties. Mixedness simulations were conducted for random heterogeneous mixtures of Nd-doped LaF3 mixtures using the Concentric Shell Model of Mixedness (CSMM). Results indicate that when the host to dopant particle size ratio is 100, multi-scale concentration variance is optimized. In order to verify results from the model, experimental methods that probe a mixture at the micro, meso, and macro scales are needed. To directly compare CSMM results experimentally, an image processing method was developed to calculate variance profiles from electron images. An in-lens (IL) secondary electron image is subtracted from the corresponding Everhart-Thornley (ET) secondary electron image in a Field-Emission Scanning Electron Microscope (FESEM) to produce two phases and pores that can be quantified with 50 nm spatial resolution. A macro was developed to quickly analyze multi-scale compositional variance from these images. Results for a 50:50 mixture of NdF3 and LaF3 agree with the computational model. The method has proven to be applicable only for mixtures with major components and specific particle morphologies, but the macro is useful for any type of imaging that produces excellent phase contrast, such as confocal microscopy. Fluorescence spectroscopy was used as an indirect method to confirm computational results for Nd-doped LaF3 mixtures. Fluorescence lifetime can be used as a quantitative method to indirectly measure chemical homogeneity when the limits of electron microscopy have been reached. Fluorescence lifetime represents the compositional fluctuations of a dopant on the nanoscale while accounting for billions of particles in a fast, non-destructive manner. The significance of this study will show how small-scale fluctuations in homogeneity limit the optimization of optical properties, which can be improved by the proper selection of particle size and mixing method.

Cell adhesion on nanotextured slippery superhydrophobic substrates.

PubMed

Di Mundo, Rosa; Nardulli, Marina; Milella, Antonella; Favia, Pietro; d'Agostino, Riccardo; Gristina, Roberto

2011-04-19

In this work, the response of Saos2 cells to polymeric surfaces with different roughness/density of nanometric dots produced by a tailored plasma-etching process has been studied. Topographical features have been evaluated by atomic force microscopy, while wetting behavior, in terms of water-surface adhesion energy, has been evaluated by measurements of drop sliding angle. Saos2 cytocompatibility has been investigated by scanning electron microscopy, fluorescent microscopy, and optical microscopy. The similarity in outer chemical composition has allowed isolation of the impact of the topographical features on cellular behavior. The results indicate that Saos2 cells respond differently to surfaces with different nanoscale topographical features, clearly showing a certain inhibition in cell adhesion when the nanoscale is particularly small. This effect appears to be attenuated in surfaces with relatively bigger nanofeatures, though these express a more pronounced slippery/dry wetting character. © 2011 American Chemical Society

Fabrication and optical characterization of imaging fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2005-09-15

In this paper, we present a technique for fabricating arrays containing a density at least 90 times higher than previously published. Specifically, we discuss the fabrication of two imaging fiber-based nanoarrays, one with 700nm features, another with 300nm features. With arrays containing up to 4.5x10(6) array elements/mm(2), these nanoarrays have an ultra-high packing density. A straightforward etching protocol is used to create nanowells into which beads can be deposited. These beads comprise the sensing elements of the nanoarray. Deposition of the nanobeads into the nanowells using two techniques is described. The surface characteristics of the etched arrays are examined with atomic force microscopy and scanning electron microscopy. Fluorescence microscopy was used to observe the arrays. The 300nm array features and the 500nm center-to-center distance approach the minimum feature sizes viewable using conventional light microscopy.

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

PubMed

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae).

PubMed

Caissard, Jean-Claude; Olivier, Thomas; Delbecque, Claire; Palle, Sabine; Garry, Pierre-Philippe; Audran, Arthur; Valot, Nadine; Moja, Sandrine; Nicolé, Florence; Magnard, Jean-Louis; Legrand, Sylvain; Baudino, Sylvie; Jullien, Frédéric

2012-01-01

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.

Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)

PubMed Central

Caissard, Jean-Claude; Olivier, Thomas; Delbecque, Claire; Palle, Sabine; Garry, Pierre-Philippe; Audran, Arthur; Valot, Nadine; Moja, Sandrine; Nicolé, Florence; Magnard, Jean-Louis; Legrand, Sylvain; Baudino, Sylvie; Jullien, Frédéric

2012-01-01

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces. PMID:23133579

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

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

PubMed

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-11-01

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. 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 G 0 /G 1 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. 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. 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. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

Cell Uptake and Validation of Novel PECs for Biomedical Applications.

PubMed

Palamà, Ilaria E; Musarò, Mariarosaria; Coluccia, Addolorata M L; D'Amone, Stefania; Gigli, Giuseppe

2011-01-01

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability.

Cell Uptake and Validation of Novel PECs for Biomedical Applications

PubMed Central

Palamà, Ilaria E.; Musarò, Mariarosaria; Coluccia, Addolorata M. L.; D'Amone, Stefania; Gigli, Giuseppe

2011-01-01

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability. PMID:21876815

Multimodal Nonlinear Optical Microscopy

PubMed Central

Yue, Shuhua; Slipchenko, Mikhail N.; Cheng, Ji-Xin

2013-01-01

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed. PMID:24353747

High-Resolution Detector For X-Ray Diffraction

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Withrow, William K.; Pusey, Marc L.; Yost, Vaughn H.

1988-01-01

Proposed x-ray-sensitive imaging detector offers superior spatial resolution, counting-rate capacity, and dynamic range. Instrument based on laser-stimulated luminescence and reusable x-ray-sensitive film. Detector scans x-ray film line by line. Extracts latent image in film and simultaneously erases film for reuse. Used primarily for protein crystallography. Principle adapted to imaging detectors for electron microscopy and fluorescence spectroscopy and general use in astronomy, engineering, and medicine.

Labeling tetracysteine-tagged proteins with biarsenical dyes for live cell imaging.

PubMed

Gaietta, Guido M; Deerinck, Thomas J; Ellisman, Mark H

2011-01-01

Correlation of real-time or time-lapse light microscopy (LM) with electron microscopy (EM) of cells can be performed with biarsenical dyes. These dyes fluorescently label tetracysteine-tagged proteins so that they can be imaged with LM and, upon fluorescent photoconversion of 3,3'-diaminobenzidine tetrahydrochloride (DAB), with EM as well. In the following protocol, cells expressing tetracysteine-tagged proteins are labeled for 1 h with biarsenical dyes. The volumes indicated are for a single 30-mm culture dish containing 2 mL of labeling medium. Scale the suggested volumes up or down depending upon the size of the culture dish used in the labeling. The same procedure can be adapted for longer labeling times by lowering the amount of dye used to 50-100 nM; however, the amount of the competing dithiol EDT is maintained at 10-20 μM. Longer labeling times often produce higher signal-to-noise ratios and cause less trauma to the treated cells prior to imaging.

Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

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

Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.

2014-12-15

Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each ofmore » which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes« less

Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks.

PubMed

Que, Emily L; Bleher, Reiner; Duncan, Francesca E; Kong, Betty Y; Gleber, Sophie C; Vogt, Stefan; Chen, Si; Garwin, Seth A; Bayer, Amanda R; Dravid, Vinayak P; Woodruff, Teresa K; O'Halloran, Thomas V

2015-02-01

Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.

In vivo imaging of kidney glomeruli transplanted into the anterior chamber of the mouse eye

PubMed Central

Kistler, Andreas D.; Caicedo, Alejandro; Abdulreda, Midhat H.; Faul, Christian; Kerjaschki, Dontscho; Berggren, Per-Olof; Reiser, Jochen; Fornoni, Alessia

2014-01-01

Multiphoton microscopy enables live imaging of the renal glomerulus. However, repeated in vivo imaging of the same glomerulus over extended periods of time and the study of glomerular function independent of parietal epithelial and proximal tubular cell effects has not been possible so far. Here, we report a novel approach for non-invasive imaging of acapsular glomeruli transplanted into the anterior chamber of the mouse eye. After microinjection, glomeruli were capable of engrafting on the highly vascularized iris. Glomerular structure was preserved, as demonstrated by podocyte specific expression of cyan fluorescent protein and by electron microscopy. Injection of fluorescence-labeled dextrans of various molecular weights allowed visualization of glomerular filtration and revealed leakage of 70 kDa dextran in an inducible model of proteinuria. Our findings demonstrate functionality and long-term survival of glomeruli devoid of Bowman's capsule and provide a novel approach for non-invasive longitudinal in vivo study of glomerular physiology and pathophysiology. PMID:24464028

Pleomorphism and Viability of the Lyme Disease Pathogen Borrelia burgdorferi Exposed to Physiological Stress Conditions: A Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy Study.

PubMed

Vancová, Marie; Rudenko, Nataliia; Vaněček, Jiří; Golovchenko, Maryna; Strnad, Martin; Rego, Ryan O M; Tichá, Lucie; Grubhoffer, Libor; Nebesářová, Jana

2017-01-01

To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.

Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

PubMed Central

Lu, Jinjin; Zheng, Xiufen; Li, Fan; Yu, Yang; Chen, Zhong; Liu, Zheng; Wang, Zhihua; Xu, Hua; Yang, Weimin

2017-01-01

Intercellular transfer of organelles via tunneling nanotubes (TNTs) is a novel means of cell-to-cell communication. Here we demonstrate the existence of TNTs between co-cultured RT4 and T24 bladder cancer cells using light microscopy, fluorescence imaging, and scanning electron microscopy (SEM). Spontaneous unidirectional transfer of mitochondria from T24 to RT4 cells was detected using fluorescence imaging and flow cytometry. The distribution of mitochondria migrated from T24 cells was in good agreement with the original mitochondria in RT4 cells, which may imply mitochondrial fusion. We detected cytoskeleton reconstruction in RT4-Mito-T24 cells by observing F-actin redistribution. Akt, mTOR, and their downstream mediators were activated and increased. The resultant increase in the invasiveness of bladder cancer cells was detected in vitro and in vivo. These data indicate that TNTs promote intercellular mitochondrial transfer between heterogeneous cells, followed by an increase in the invasiveness of bladder cancer cells. PMID:28107184

Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

PubMed Central

Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.; Kong, Betty Y.; Gleber, Sophie C.; Vogt, Stefan; Chen, Si; Garwin, Seth A.; Bayer, Amanda R.; Dravid, Vinayak; Woodruff, Teresa K.; O’Halloran, Thomas V.

2015-01-01

Fertilization of a mammalian egg induces a series of ‘zinc sparks’ that are necessary for inducing the egg-to-embryo transition. Despite the importance of these zinc efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches to resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy dispersive spectroscopy, X-ray fluorescence microscopy, and 3D elemental tomography for high resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 106 zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes. PMID:25615666

Target binding influences permeability in aptamer-polyelectrolyte microcapsules.

PubMed

Sultan, Yasir; DeRosa, Maria C

2011-05-09

Aptamer-polyelectrolyte microcapsules are prepared for potential use as triggered delivery vehicles and microreactors. The hollow microcapsules are prepared from the sulforhodamine B aptamer and the polyelectrolytes poly(allylamine hydrochloride) and poly(sodium 4-styrene-sulfonate), using layer-by-layer (LbL) film deposition templated on a sacrificial CaCO(3) spherical core. Scanning electron microscopy and confocal microscopy confirm the formation of spherical CaCO(3) cores and LbL-aptamer microcapsules. Colocalization studies with fluorescently-tagged aptamer and sulforhodamine B verify the ability of the aptamer to recognize its cognate target in the presence of the K(+) ions that are required for its characteristic G-quadruplex formation. Fluorescence recovery after photobleaching studies confirms a significant difference in the permeability of the aptamer-polyelectrolyte microcapsules for the sulforhodamine B dye target compared to control microcapsules prepared with a random oligonucleotide. These results suggest that aptamer-based 'smart' responsive films and microcapsules could be applied to problems of catalysis and controlled release. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

NASA Technical Reports Server (NTRS)

Szmacinski, Henryk

2003-01-01

Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report.

New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64.

PubMed

Nishihara, E; Shimmen, T; Sonobe, S

2007-01-01

The contractile vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

PubMed Central

Mayor, S; Sabharanjak, S; Maxfield, F R

1998-01-01

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins. PMID:9707422

Chondroptosis in Alkaptonuric Cartilage

PubMed Central

Millucci, Lia; Giorgetti, Giovanna; Viti, Cecilia; Ghezzi, Lorenzo; Gambassi, Silvia; Braconi, Daniela; Marzocchi, Barbara; Paffetti, Alessandro; Lupetti, Pietro; Bernardini, Giulia; Orlandini, Maurizio

2015-01-01

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. 230: 1148–1157, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:25336110

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.

Synthesis of novel β-cyclodextrin functionalized S, N codoped carbon dots for selective detection of testosterone.

PubMed

Luo, Mai; Hua, Yifan; Liang, Yiran; Han, Jiajun; Liu, Donghui; Zhao, Wenting; Wang, Peng

2017-12-15

A novel functionalized carbon dot has been synthesized by covalently linking β-cyclodextrin to the surface of N, S codoped carbon dots (β-CD-CDs). The characterization was confirmed by transmission electron microscopy, X-ray photoelectron spectroscopy, infrared spectra, ultraviolet-visible, and fluorescence emission spectra. On the basis of this carbon dot and (ferrocenylmethyl) trimethylammonium iodide (Fc + ), a photo-induced electron transfer (PET) fluorescent probe system was developed to determine the concentration of testosterone in water and identify testosterone in cell by fluorescence imaging as a visible biomarker. Under the optimum condition, the fluorescent intensity of the probe system linearly responded to the concentration of testosterone from 0μM to 280μM and the limit of detection was 0.51μM. This probe system also performed well at determining testosterone in groundwater with average recoveries of testosterone ranging from 96% to 107% at spiking levels of 0.5-100μM, and the relative standard deviation remained below 13%, which provided a reliable, rapid and easy method to determine testosterone in environmental water. Furthermore, the low cytotoxicity, high anti-interference ability, and excellent biocompatibility of β-CD-CDs made this probe system successfully used in cell fluorescence imaging to monitor levels of testosterone in the cytoplasm of cells with a promising application value in medical research. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae.

PubMed

Khamhaengpol, Arunrat; Siri, Sineenat

2016-10-01

Alternative to crude plant extracts, a crude protein extract derived from animal cells is one of the potential sources of biomolecules for mediating a reduction of silver ions and a formation of silver nanoparticles (AgNPs) under a mild condition, which very few works have been reported. This work demonstrated a use of the protein extract of weaver ant larvae as a bio-facilitator for a simple, green synthesis of AgNPs under fluorescent light at room temperature. The protein extract of weaver ant larvae exhibited the reducing and antioxidant activities, which assisted a formation of AgNPs in the reaction containing only silver nitrate under light exposure. Transmission electron microscopy images revealed the dispersed, spherical AgNPs with an average size of 7.87±2.54nm. The maximum surface plasmon resonance (SPR) band of the synthesized AgNPs was at 435nm. The energy-dispersive X-ray analysis revealed that silver was a major element of the particles. The identity of AgNPs was confirmed by X-ray diffraction pattern, selected area electron diffraction and high resolution transmission electron microscopy analyses, which demonstrated the planes of face centered cubic silver. The synthesized AgNPs showed antibacterial activity against both Escherichia coli and Staphylococcus aureus with the minimum bactericidal concentration (MBC) values equally at 250μg/ml, suggesting their potential application as an effective antibacterial agent. Copyright © 2016 Elsevier B.V. All rights reserved.

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques

PubMed Central

Ostrowski, Anja; Nordmeyer, Daniel; Boreham, Alexander; Holzhausen, Cornelia; Mundhenk, Lars; Graf, Christina; Meinke, Martina C; Vogt, Annika; Hadam, Sabrina; Lademann, Jürgen; Rühl, Eckart; Alexiev, Ulrike

2015-01-01

Summary The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties. PMID:25671170

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

Coherent Anti-Stokes Raman Scattering Spectroscopy of Single Molecules in Solution

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

Sunney Xie, Wei Min, Chris Freudiger, Sijia Lu

2012-01-18

During this funding period, we have developed two breakthrough techniques. The first is stimulated Raman scattering microscopy, providing label-free chemical contrast for chemical and biomedical imaging based on vibrational spectroscopy. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. We developed a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-freemore » and readily interpretable chemical contrast. We demonstrated a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis. This technology offers exciting prospect for medical imaging. The second technology we developed is stimulated emission microscopy. Many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. We use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, as a new contrast mechanism for optical microscopy. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distribu- tions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging. Although we did not accomplish the original goal of detecting single-molecule by CARS, our quest for high sensitivity of nonlinear optical microscopy paid off in providing the two brand new enabling technologies. Both techniques were greatly benefited from the use of high frequency modulation for microscopy, which led to orders of magnitude increase in sensitivity. Extensive efforts have been made on optics and electronics to accomplish these breakthroughs.« less

Characterization of As-polluted soils by laboratory X-ray-based techniques coupled with sequential extractions and electron microscopy: the case of Crocette gold mine in the Monte Rosa mining district (Italy).

PubMed

Allegretta, Ignazio; Porfido, Carlo; Martin, Maria; Barberis, Elisabetta; Terzano, Roberto; Spagnuolo, Matteo

2018-06-24

Arsenic concentration and distribution were studied by combining laboratory X-ray-based techniques (wavelength dispersive X-ray fluorescence (WDXRF), micro X-ray fluorescence (μXRF), and X-ray powder diffraction (XRPD)), field emission scanning electron microscopy equipped with microanalysis (FE-SEM-EDX), and sequential extraction procedure (SEP) coupled to total reflection X-ray fluorescence (TXRF) analysis. This approach was applied to three contaminated soils and one mine tailing collected near the gold extraction plant at the Crocette gold mine (Macugnaga, VB) in the Monte Rosa mining district (Piedmont, Italy). Arsenic (As) concentration, measured with WDXRF, ranged from 145 to 40,200 mg/kg. XRPD analysis evidenced the presence of jarosite and the absence of any As-bearing mineral, suggesting a high weathering grade and strong oxidative conditions. However, small domains of Fe arsenate were identified by combining μXRF with FE-SEM-EDX. SEP results revealed that As was mainly associated to amorphous Fe oxides/hydroxides or hydroxysulfates (50-80%) and the combination of XRPD and FE-SEM-EDX suggested that this phase could be attributed to schwertmannite. On the basis of the reported results, As is scarcely mobile, even if a consistent As fraction (1-3 g As/kg of soil) is still potentially mobilizable. In general, the proposed combination of laboratory X-ray techniques could be successfully employed to unravel environmental issues related to metal(loid) pollution in soil and sediments.

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.

Highly efficient red fluorescent organic light-emitting diodes by sorbitol-doped PEDOT:PSS

NASA Astrophysics Data System (ADS)

Zheng, Yan-Qiong; Yu, Jun-Le; Wang, Chao; Yang, Fang; Wei, Bin; Zhang, Jian-Hua; Zeng, Cheng-Hui; Yang, Yang

2018-06-01

This work shows a promising approach to improve device performance by optimizing the electron transport and hole injection layers for tetraphenyldibenzoperiflanthene (DBP):rubrene-based red fluorescent organic light-emitting diodes (OLEDs). We compared the effect of two electron transport layers (ETLs), and found that the rubrene/bathophenanthroline (Bphen) ETL-based OLED showed a much higher external quantum efficiency (EQE) (4.67%) than the Alq3 ETL-based OLED (EQE of 3.08%). The doping ratio of DBP in rubrene was tuned from 1.0 wt% to 4.5 wt%, and the 1.5 wt%-DBP:rubrene-based OLED demonstrated the highest EQE of 5.24% and lowest turn-on voltage of 2.2 V. Atomic force microscopy images indicated that 1.5 wt% DBP-doped rubrene film exhibited a regular strip shape, and this regular surface was favorable to the hole and electron recombination in the emitting layer. Finally, the sorbitol-doped poly(3, 4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) was used to further improve the EQE; doping with 6 wt% sorbitol achieved the highest current efficiency of 7.03 cd A‑1 and an EQE of 7.50%. The significantly enhanced performance implies that the hole injection is a limiting factor for DBP:rubrene-based red fluorescent OLEDs.

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

Identification of Foreign Particles in Human Tissues using Raman Microscopy.

PubMed

Campion, Alan; Smith, Kenneth J; Fedulov, Alexey V; Gregory, David; Fan, Yuwei; Godleski, John J

2018-06-12

The precise identification of foreign particles in tissue for patient care and research has been studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction. The goal of this study was to unambiguously identify particles in tissues using a combina-tion of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicron spatial resolution. We designed a model system of stained and unstained cells that contained birefringent talc particles, and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra ob-tained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely as-signed to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micron in diameter. Among commonly used coverslip mounting media, Cytoseal 60 is recommended; Permount was unacceptable due to intense background interference. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251

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

Non-Noble Metal Oxide Catalysts for Methane Catalytic Combustion: Sonochemical Synthesis and Characterisation

PubMed Central

Jędrzejczyk, Roman J.; Dziedzicka, Anna; Kuterasiński, Łukasz; Sitarz, Maciej

2017-01-01

The aim of this study was to obtain nanocrystalline mixed metal-oxide–ZrO2 catalysts via a sonochemically-induced preparation method. The effect of a stabiliser’s addition on the catalyst parameters was investigated by several characterisation methods including X-ray Diffraction (XRD), nitrogen adsorption, X-ray fluorescence (XRF), scanning electron microscopy (SEM) equipped with energy dispersive X-ray spectrometer (EDS), transmission electron microscopy (TEM) and µRaman. The sonochemical preparation method allowed us to manufacture the catalysts with uniformly dispersed metal-oxide nanoparticles at the support surface. The catalytic activity was tested in a methane combustion reaction. The activity of the catalysts prepared by the sonochemical method was higher than that of the reference catalysts prepared by the incipient wetness method without ultrasonic irradiation. The cobalt and chromium mixed zirconia catalysts revealed their high activities, which are comparable with those presented in the literature. PMID:28686190

Atomic level characterization of cadmium selenide nanocrystal systems using atomic number contrast scanning transmission electron microscopy and Rutherford backscattering spectroscopy

NASA Astrophysics Data System (ADS)

McBride, James R.

This project involved the characterization of CdSe nanocrystals. Through the use of Atomic Number Contrast Scanning Transmission Electron Microscopy (Z-STEM) and Rutherford Backscattering Spectroscopy (RBS), atomic level structure and chemical information was obtained. Specifically, CdSe nanocrystals produced using a mixture of hexadecylamine (HDA) and trioctylphosphine oxide (TOPO) were determined to be spherical compared to nanocrystals produced in TOPO only, which had elongated (101) facets. Additionally, the first Z-STEM images of CdSe/ZnS core/shell nanocrystals were obtained. From these images, the growth mechanism of the ZnS shell was determined and the existence of non-fluorescent ZnS particles was confirmed. Through collaboration with Quantum Dot Corp., core/shell nanocrystals with near unity quantum yield were developed. These core/shell nanocrystals included a US intermediate layer to improve shell coverage.

Synthesis procedure optimization and characterization of europium (III) tungstate nanoparticles

NASA Astrophysics Data System (ADS)

Rahimi-Nasrabadi, Mehdi; Pourmortazavi, Seied Mahdi; Ganjali, Mohammad Reza; Reza Banan, Ali; Ahmadi, Farhad

2014-09-01

Taguchi robust design as a statistical method was applied for the optimization of process parameters in order to tunable, facile and fast synthesis of europium (III) tungstate nanoparticles. Europium (III) tungstate nanoparticles were synthesized by a chemical precipitation reaction involving direct addition of europium ion aqueous solution to the tungstate reagent solved in an aqueous medium. Effects of some synthesis procedure variables on the particle size of europium (III) tungstate nanoparticles were studied. Analysis of variance showed the importance of controlling tungstate concentration, cation feeding flow rate and temperature during preparation of europium (III) tungstate nanoparticles by the proposed chemical precipitation reaction. Finally, europium (III) tungstate nanoparticles were synthesized at the optimum conditions of the proposed method. The morphology and chemical composition of the prepared nano-material were characterized by means of X-ray diffraction, scanning electron microscopy, transmission electron microscopy, FT-IR spectroscopy and fluorescence.

Reversible sol-to-gel transformation of uracil gelators: specific colorimetric and fluorimetric sensor for fluoride ions.

PubMed

Xing, Ling-Bao; Yang, Bing; Wang, Xiao-Jun; Wang, Jiu-Ju; Chen, Bin; Wu, Qianhong; Peng, Hui-Xing; Zhang, Li-Ping; Tung, Chen-Ho; Wu, Li-Zhu

2013-03-05

A new type of anthracene organogelator based on uracil was obtained using organic aromatic solvents, cyclohexane, DMSO, ethanol, and ethyl acetate. It was further characterized by field-emission scanning electron microscopy and transmission electron microscopy. Specifically, the resulting organogels were demonstrated to be promising colorimetric and fluorescent sensors toward fluoride ions with high sensitivity and selectivity, accompanying the disruption of the gelators. Spectroscopic study and (1)H NMR titration experiment revealed that the deprotonation of the hydrogen atom on the N position of uracil moiety by fluoride ions is responsible for the recognition events, evidenced by immediate transformation from the sol phase to the gel state upon adding a small amount of a proton solvent, methanol. The process is reversible, with zero loss in sensing activity and sol-to-gel transformation ability even after five runs.

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

Myllys, Markko; Ruokolainen, Visa; Aho, Vesa

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gapsmore » frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.« less

Physicochemical properties of micelles of poly(styrene-b-[3-(methacryloylamino)propyl]trimethylammonium chloride-b-ethylene oxide) in aqueous solutions.

PubMed

Liu, Jingjing; Liu, Dian; Yokoyama, Yuuichi; Yusa, Shin-Ichi; Nakashima, Kenichi

2009-01-20

Polymeric micelles from a new triblock copolymer, polystyrene-block-poly[(3-(methacryloylamino)propyl)trimethylammonium chloride]-block-poly(ethylene oxide) (PS-b-PMAPTAC-b-PEO), were prepared in aqueous solutions and characterized by various techniques including dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence spectroscopy. The micelle consists of a PS core, PMAPTAC shell, and PEO corona. It was revealed by SEM and DLS measurements that the micelles have a spherical structure with a hydrodynamic diameter about 75 nm. The addition of tungstate to the micellar solution caused a morphological change in the micelles from extended to shrunken spheres, which can be attributed to the fact that electrostatic repulsion among the cationic PMAPTAC blocks is canceled by the negative charge of the bound tungstate ions. Effective incorporation of tungstate ions into the micelles were confirmed by TEM and zeta-potential measurements.

Nano-crystalline hydroxyapatite bio-mineral for the treatment of strontium from aqueous solutions.

PubMed

Handley-Sidhu, Stephanie; Renshaw, Joanna C; Yong, Ping; Kerley, Robert; Macaskie, Lynne E

2011-01-01

Hydroxyapatites were analysed using electron microscopy, X-ray diffraction (XRD) and X-ray fluorescence (XRF) analysis. Examination of a bacterially produced hydroxyapatite (Bio-HA) by scanning electron microscopy showed agglomerated nano-sized particles; XRD analysis confirmed that the Bio-HA was hydroxyapatite, with an organic matter content of 7.6%; XRF analysis gave a Ca/P ratio of 1.55, also indicative of HA. The size of the Bio-HA crystals was calculated as ~25 nm from XRD data using the Scherrer equation, whereas Comm-HA powder size was measured as ≤ 50 μm. The nano-crystalline Bio-HA was ~7 times more efficient in removing Sr(2+) from synthetic groundwater than Comm-HA. Dissolution of HA as indicated by the release of phosphate into the solution phase was higher in the Comm-HA than the Bio-HA, indicating a more stable biomaterial which has a potential for the remediation of contaminated sites.

Herpes simplex virus 1 induces egress channels through marginalized host chromatin

DOE PAGES

Myllys, Markko; Ruokolainen, Visa; Aho, Vesa; ...

2016-06-28

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gapsmore » frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.« less

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

Differential polarization nonlinear optical microscopy with adaptive optics controlled multiplexed beams.

PubMed

Samim, Masood; Sandkuijl, Daaf; Tretyakov, Ian; Cisek, Richard; Barzda, Virginijus

2013-09-09

Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Stability and degradation of organic photovoltaics fabricated, aged, and characterized by the ISOS 3 inter-laboratory collaboration

NASA Astrophysics Data System (ADS)

Tanenbaum, David M.; Hermenau, Martin; Voroshazi, Eszter; Lloyd, Matthew T.; Galagan, Yulia; Zimmermann, Birger; Hösel, Markus; Dam, Henrik F.; Jørgensen, Mikkel; Gevorgyan, Suren; Kudret, Suleyman; Maes, Wouter; Lutsen, Laurence; Vanderzande, Dirk; Würfel, Uli; Andriessen, Ronn; Rösch, Roland; Hoppe, Harald; Lira-Cantu, Monica; Teran-Escobar, Gerardo; Dupuis, Aurélie; Bussière, Pierre-Olivier; Rivaton, Agnès.; Uzunoglu, Gülsah Y.; Germack, David; Andreasen, Birgitta; Madsen, Morten V.; Norrman, Kion; Bundgaard, Eva; Krebs, Frederik C.

2012-09-01

Seven distinct sets (n >= 12) of state of the art organic photovoltaic devices were prepared by leading research laboratories in a collaboration planned at the Third International Summit on Organic Photovoltaic Stability (ISOS-3). All devices were shipped to DTU and characterized simultaneously up to 1830 h in accordance with established ISOS-3 protocols under three distinct illumination conditions: accelerated full sun simulation; low level indoor fluorescent lighting; and dark storage with daily measurement under full sun simulation. Three nominally identical devices were used in each experiment both to provide an assessment of the homogeneity of the samples and to distribute samples for a variety of post soaking analytical measurements at six distinct laboratories enabling comparison at various stages in the degradation of the devices. Characterization includes current-voltage curves, light beam induced current (LBIC) imaging, dark lock-in thermography (DLIT), photoluminescence (PL), electroluminescence (EL), in situ incident photon-to-electron conversion efficiency (IPCE), time of flight secondary ion mass spectrometry (TOF-SIMS), cross sectional electron microscopy (SEM), UV visible spectroscopy, fluorescence microscopy, and atomic force microscopy (AFM). Over 100 devices with more than 300 cells were used in the study. We present here design of the device sets, results both on individual devices and uniformity of device sets from the wide range of characterization methods applied at different stages of aging under the three illumination conditions. We will discuss how these data can help elucidate the degradation mechanisms as well as the benefits and challenges associated with the unprecedented size of the collaboration.

Cholecystolithiasis is associated with Clonorchis sinensis infection.

PubMed

Qiao, Tie; Ma, Rui-hong; Luo, Xiao-bing; Luo, Zhen-liang; Zheng, Pei-ming

2012-01-01

The objective of this study was to analyze gallbladder stones for direct evidence of a relationship between Clonorchis sinensis infection and gallbladder stones formation. We investigated one hundred eighty-three gallbladder stones for the presence of Clonorchis sinensis eggs using microscopy, and analyzed their composition using Fourier transform infrared spectroscopy. We confirmed the presence of Clonorchis sinensis eggs in the gallbladder stones using real-time fluorescent PCR and scanning electron microscopy. Clonorchis sinensis eggs were detected in 122 of 183 gallbladder stones based on morphologic characteristics and results from real-time fluorescent PCR. The proportion of pigment stones, cholesterol stones and mixed gallstones in the egg-positive stones was 79.5% (97/122), 3.3% (4/122) and 17.2% (21/122), respectively, while 29.5% (18/61), 31.1% (19/61) and 39.3% (24/61) in the egg-negative stones. The proportion of pigment stone in the Clonorchis sinensis egg-positive stones was higher than in egg-negative stones (P<0.0001). In the 30 egg-positive stones examined by scanning electron microscopy, dozens or even hundreds of Clonorchis sinensis eggs were visible (×400) showing a distinct morphology. Many eggs were wrapped with surrounding particles, and in some, muskmelon wrinkles was seen on the surface of the eggs. Also visible were pieces of texture shed from some of the eggs. Some eggs were depressed or without operculum while most eggs were adhered to or wrapped with amorphous particles or mucoid matter (×3000). Clonorchis sinensis eggs were detected in the gallbladder stones which suggests an association between Clonorchis sinensis infection and gallbladder stones formation, especially pigment stones.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

PubMed

Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

2015-04-01

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

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.

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.

Monosynaptic convergence of chorda tympani and glossopharyngeal afferents onto ascending relay neurons in the nucleus of the solitary tract: A high-resolution confocal and correlative electron microscopy approach

PubMed Central

Corson, James A.; Erisir, Alev

2014-01-01

While physiological studies suggested convergence of chorda tympani and glossopharyngeal afferent axons onto single neurons of the rostral nucleus of the solitary tract (rNTS), anatomical evidence has been elusive. The current study uses high-magnification confocal microscopy to identify putative synaptic contacts from afferent fibers of the two nerves onto individual projection neurons. Imaged tissue is re-visualized with electron microscopy, confirming that overlapping fluorescent signals in confocal z-stacks accurately identify appositions between labeled terminal and dendrite pairs. Monte Carlo modeling reveals that the probability of overlapping fluorophores is stochastically unrelated to the density of afferent label suggesting that convergent innervation in the rNTS is selective rather than opportunistic. Putative synaptic contacts from each nerve are often compartmentalized onto dendrite segments of convergently innervated neurons. These results have important implications for orosensory processing in the rNTS, and the techniques presented here have applications in investigations of neural microcircuitry with an emphasis on innervation patterning. PMID:23640852

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".

Controlled transport of latex beads through vertically aligned carbon nanofiber membranes

NASA Astrophysics Data System (ADS)

Zhang, L.; Melechko, A. V.; Merkulov, V. I.; Guillorn, M. A.; Simpson, M. L.; Lowndes, D. H.; Doktycz, M. J.

2002-07-01

Stripes of vertically aligned carbon nanofibers (VACNFs) have been used to form membranes for size selectively controlling the transport of latex beads. Fluidic structures were created in poly(dimethylsiloxane) (PDMS) and interfaced to the VACNF structures for characterization of the membrane pore size. Solutions of fluorescently labeled latex beads were introduced into the PDMS channels and characterized by fluorescence and scanning electron microscopy. Results show that the beads size selectively pass through the nanofiber barriers and the size restriction limit correlates with the interfiber spacing. The results suggest that altering VACNF array density can alter fractionation properties of the membrane. Such membranes may be useful for molecular sorting and for mimicking the properties of natural membranes.

ZnO:Gd nanocrystals for fluorescent applications

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

Divya, N. K., E-mail: divyank90@gmail.com; Pradyumnan, P. P.

2016-05-23

Gadolinium doped ZnO crystals within the solubility limit of gadolinium in ZnO matrix were prepared by solid state reaction technique. The method is relatively less expense and enables the production in large scale. The samples were characterised by X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), UV/Vis diffuse reflectance spectroscopy and photoluminescence techniques. Fluorescent property studies of gadolinium doped ZnO at room temperature show enhanced visible light emission due to the defects and oxygen vacancies produced via doping. This work reports the impact of gadolinium doping in the structural, optical and luminescent properties of ZnO inmore » detail.« less

A quantum dot-spore nanocomposite pH sensor.

PubMed

Zhang, Xingya; Li, Zheng; Zhou, Tao; Zhou, Qian; Zeng, Zhiming; Xu, Xiangdong; Hu, Yonggang

2016-04-01

A new quantum dot (QD)-based pH sensor design is investigated. The sensor is synthesized based on the self-assembly of green QDs onto treated spores to form QD@spore nanocomposites. The nanocomposites are characterized using laser scanning confocal microscopy, transmission electron microscope, and fluorescence spectroscopy, among others. Fluorescence measurements showed that these nanocomposites are sensitive to pH in a broad pH range of 5.0-10.0. The developed pH sensors have been satisfactorily applied for pH estimation of real samples and are comparable with those of the commercial assay method, indicating the potential practical application of the pH sensors. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Visualizing different uranium oxidation states during the surface alteration of uraninite and uranium tetrachloride.

PubMed

Grossmann, Kay; Arnold, Thuro; Steudtner, Robin; Weiss, Stefan; Bernhard, Gert

2009-08-01

Low-temperature alteration reactions on uranium phases may lead to the mobilization of uranium and thereby poses a potential threat to humans living close to uranium-contaminated sites. In this study, the surface alteration of uraninite (UO(2)) and uranium tetrachloride (UCl(4)) in air atmosphere was studied by confocal laser scanning microscopy (CLSM) and laser-induced fluorescence spectroscopy using an excitation wavelength of 408 nm. It was found that within minutes the oxidation state on the surface of the uraninite and the uranium tetrachloride changed. During the surface alteration process U(IV) atoms on the uraninite and uranium tetrachloride surface became stepwise oxidized by a one-electron step at first to U(V) and then further to U(VI). These observed changes in the oxidation states of the uraninite surface were microscopically visualized and spectroscopically identified on the basis of their fluorescence emission signal. A fluorescence signal in the wavelength range of 415-475 nm was indicative for metastable uranium(V), and a fluorescence signal in the range of 480-560 nm was identified as uranium(VI). In addition, the oxidation process of tetravalent uranium in aqueous solution at pH 0.3 was visualized by CLSM and U(V) was fluorescence spectroscopically identified. The combination of microscopy and fluorescence spectroscopy provided a very convincing visualization of the brief presence of U(V) as a metastable reaction intermediate and of the simultaneous coexistence of the three states U(IV), U(V), and U(VI). These results have a significant importance for fundamental uranium redox chemistry and should contribute to a better understanding of the geochemical behavior of uranium in nature.

Folate receptor targeted, rare-earth oxide nanocrystals for bi-modal fluorescence and magnetic imaging of cancer cells.

PubMed

Setua, Sonali; Menon, Deepthy; Asok, Adersh; Nair, Shantikumar; Koyakutty, Manzoor

2010-02-01

Targeted cancer imaging using rare-earth oxide nanocrystals, free from heavy metals (Cd, Se, Te, Hg and Pb), showing bright red-fluorescence and magnetic resonance imaging (MRI) is presented. Y(2)O(3) nanocrystals (YO NC) doped in situ with fluorescent (Eu(3+)) and paramagnetic (Gd(3+)) impurities and conjugated with a potential cancer targeting ligand, folic acid (FA), were prepared using an all-aqueous wet-chemical process. Structural, optical and magnetic properties of these multifunctional nanocrystals were investigated by X-ray diffraction, electron microscopy, photoluminescence and magnetization studies. Highly monodisperse nanocrystals of size approximately 20 nm with cubic bixbyite crystal structure showed bright red-fluorescence when doped with Eu(3+). Co-doping with Gd(3+) and mild air drying resulted significantly enhanced fluorescence quantum efficiency of approximately 60% together with paramagnetic functionality, enabling T(1)-weighted MR contrast with approximately 5 times higher spin-lattice relaxivity compared to the clinically used Gd(3+) contrast agent. Cytotoxicity and reactive oxygen stress studies show no toxicity by YO NC in both normal and cancer cells up to higher doses of 500 microm and longer incubation time, 48h. Cancer targeting capability of FA conjugated NCs was demonstrated on folate receptor positive (FR+) human nasopharyngeal carcinoma cells (KB) with FR depressed KB (FRd) and FR negative (FR-) lung cancer cells A549 as controls. Fluorescence microscopy and flow-cytometry data show highly specific binding and cellular uptake of large concentration of FA conjugated NCs on FR+ve cells compared to the controls. Thus, the present study reveals, unique bi-modal contrast imaging capability, non-toxicity and cancer targeting capability of multiple impurities doped rare-earth oxide nanocrystals that can find promising application in molecular imaging.

Intracellular distribution and stability of a luminescent rhenium(I) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging

DOE PAGES

Wedding, Jason L.; Harris, Hugh H.; Bader, Christie A.; ...

2016-11-23

Optical fluorescence microscopy was used in conjunction with X-ray fluorescence microscopy to monitor the stability and intracellular distribution of the luminescent rhenium(I) complex fac-[Re(CO) 3(phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex, in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence techniques. Furthermore, X-ray fluorescence also showed that the Re-I complex disrupted the homeostasis of some biologically relevant elements,more » such as chlorine, potassium and zinc.« 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.

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.

Correlative Light and Electron Microscopy (CLEM) and its applications in infectious disease

DTIC Science & Technology

2016-05-20

Martin, Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a...diagnostic tool for functionally related structural biological studies. CLEM is particularly useful in infectious disease research where there is need to...25KD, there are critiques and concerns that the size of the tag and oligomers formation may affect the function of the target protein and introduce

Composition of prehistoric rock-painting pigments from Egypt (Gilf Kébir area).

PubMed

Darchuk, L; Rotondo, G Gatto; Swaenen, M; Worobiec, A; Tsybrii, Z; Makarovska, Y; Van Grieken, R

2011-12-01

The composition of rock-painting pigments from Egypt (Gilf Kebia area) has been analyzed by means of molecular spectroscopy such as Fourier transform infrared and micro-Raman spectroscopy and scanning electron microscopy coupled to an energy dispersive X-ray spectrometer and X-ray fluorescence analysis. Red and yellow pigments were recognized as red and yellow ochre with additional rutile. Copyright © 2011 Elsevier B.V. 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.

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays

PubMed Central

Rich, Ryan; Li, Ji; Fudala, Rafal; Gryczynski, Zygmunt; Gryczynski, Ignacy; Mandecki, Wlodek

2012-01-01

Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid-based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments we found the depth of the polymer matrix to be 1–2 µm. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2 µm. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering (SERS) is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform. PMID:22960796

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.

Preparation of fluorescent nanodiamond suspensions using bead-assisted ultrasonic disintegration

NASA Astrophysics Data System (ADS)

Głowacki, Maciej J.; Gardas, Mateusz; Ficek, Mateusz; Sawczak, Mirosław; Bogdanowicz, Robert

2017-08-01

Nitrogen-vacancy (N-V) centers are the most widely studied crystallographic defect in the diamond lattice since their presence causes strong and stable fluorescence. The negative charge state of the defect (NV-) is especially desired because of its potential for quantum information processing. In this study, fluorescent suspensions of diamond particles have been produced by microbead-assisted ultrasonic disintegration of commercially obtained diamond powder containing N-V color centers. Zirconium dioxide ZrO2 was chosen as an abrasive and a mixture of deionized water and dimethyl sulfoxide (DMSO) was used as a solvent. Raman spectrum of the starting material has been obtained and the resulting liquids have been measured in terms of photoluminescence. Moreover, thin layer of the diamond particles has been deposited on a silicon substrate and examined using scanning electron microscopy (SEM). During the course of the experiment a new method, which uses sodium chloride NaCl as an abrasive, has been proposed. The results of fluorescence measurements of the suspension prepared using this technique are highly promising.

Decoding of quantum dots encoded microbeads using a hyperspectral fluorescence imaging method.

PubMed

Liu, Yixi; Liu, Le; He, Yonghong; Zhu, Liang; Ma, Hui

2015-05-19

We presented a decoding method of quantum dots encoded microbeads with its fluorescence spectra using line scan hyperspectral fluorescence imaging (HFI) method. A HFI method was developed to attain both the spectra of fluorescence signal and the spatial information of the encoded microbeads. A decoding scheme was adopted to decode the spectra of multicolor microbeads acquired by the HFI system. Comparison experiments between the HFI system and the flow cytometer were conducted. The results showed that the HFI system has higher spectrum resolution; thus, more channels in spectral dimension can be used. The HFI system detection and decoding experiment with the single-stranded DNA (ssDNA) immobilized multicolor beads was done, and the result showed the efficiency of the HFI system. Surface modification of the microbeads by use of the polydopamine was characterized by the scanning electron microscopy and ssDNA immobilization was characterized by the laser confocal microscope. These results indicate that the designed HFI system can be applied to practical biological and medical applications.

Structural design of intrinsically fluorescent oxysterols.

PubMed

Nåbo, Lina J; Modzel, Maciej; Krishnan, Kathiresan; Covey, Douglas F; Fujiwara, Hideji; Ory, Daniel S; Szomek, Maria; Khandelia, Himanshu; Wüstner, Daniel; Kongsted, Jacob

2018-05-01

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging

PubMed Central

Bright, Vanessa

2011-01-01

A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by conjugation of superparamagnetic Fe3O4 nanoparticles and visible light-emitting (~600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. Synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive X-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (~800 nm) by conjugation of superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. Observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging. PMID:21597146

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

Synthesis of mesoporous Mn/TiO2 nanocomposites and investigating the photocatalytic properties in aqueous systems.

PubMed

Oseghe, Ekemena Oghenovoh; Ndungu, Patrick Gathura; Jonnalagadda, Sreekanth Babu

2015-01-01

Mesoporous 20 wt% Mn/TiO2 nanocomposites were synthesized adopting modified sol-gel method at different pH (pH = 2, 7 and 11) conditions and calcined at 400 °C. Based on the characteristics of the 20 wt% Mn/TiO2 nanocomposites synthesized at pH 11, same procedure was adopted for the synthesis of different wt% Mn/TiO2. The nanocomposite samples and their surface properties were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), mapping, inductively coupled plasma optical emission spectrometry (ICP-OES), Fourier transform infrared (FTIR), and fluorescence spectrometry. The nanocomposites existed in the anatase phase of TiO2 with no peak assigned to Mn on the diffractogram. The photocatalytic activities of the materials were evaluated by monitoring degradation of a model dye (methylene blue (MB)) in presence of visible light and ozone. The nanocomposite synthesized under neutral condition (pH = 7) exhibited the best photocatalytic activity resulting from its relatively smaller crystal size (5.98 nm) and larger pore volume (0.30 cm(3)/g). One percentage of weight Mn/TiO2 showed 100% decolouration of MB in the presence of O3 after 100 min.

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.

Extending the knowledge in histochemistry and cell biology.

PubMed

Heupel, Wolfgang-Moritz; Drenckhahn, Detlev

2010-01-01

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird's- up to worm's-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in "histo-biochemical" techniques and their manifold applications.

Encapsulation efficiency of CdSe/ZnS quantum dots by liposomes determined by thermal lens microscopy

PubMed Central

Batalla, Jessica; Cabrera, Humberto; San Martín-Martínez, Eduardo; Korte, Dorota; Calderón, Antonio; Marín, Ernesto

2015-01-01

In this study the encapsulation of core shell carboxyl CdSe/ZnS quantum dots (QDs) by phospholipids liposome complexes is presented. It makes the quantum dots water soluble and photo-stable. Fluorescence self-quenching of the QDs inside the liposomes was observed. Therefore, the thermal lens microscopy (TLM) was found to be an useful tool for measuring the encapsulation efficiency of the QDs by the liposomes, for which an optimum value of 36% was determined. The obtained limit of detection (LOD) for determining QDs concentration by TLM was 0.13 nM. Moreover, the encapsulated QDs showed no prominent cytotoxicity toward Breast cancer cells line MDA-MB-231. This study was supported by UV-visible spectroscopy, high resolution transmission electron microscopy (HRTEM) and dynamic light scattering measurements (DLS). PMID:26504640

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

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.

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation.

PubMed

Kopke, Danielle L; Broadie, Kendal

2018-05-24

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K + ] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Preparation of monolithic silica-chitin composite under extreme biomimetic conditions.

PubMed

Bazhenov, Vasilii V; Wysokowski, Marcin; Petrenko, Iaroslav; Stawski, Dawid; Sapozhnikov, Philipp; Born, René; Stelling, Allison L; Kaiser, Sabine; Jesionowski, Teofil

2015-05-01

Chitin is a widespread renewable biopolymer that is extensively distributed in the natural world. The high thermal stability of chitin provides an opportunity to develop novel inorganic-organic composites under hydrothermal synthesis conditions in vitro. For the first time, in this work we prepared monolithic silica-chitin composite under extreme biomimetic conditions (80°C and pH 1.5) using three dimensional chitinous matrices isolated from the marine sponge Aplysina cauliformis. The resulting material was studied using light and fluorescence microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy. A mechanism for the silica-chitin interaction after exposure to these hydrothermal conditions is proposed and discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Interaction of Water-Soluble CdTe Quantum Dots with Bovine Serum Albumin

PubMed Central

2011-01-01

Semiconductor nanoparticles (quantum dots) are promising fluorescent markers, but it is very little known about interaction of quantum dots with biological molecules. In this study, interaction of CdTe quantum dots coated with thioglycolic acid (TGA) with bovine serum albumin was investigated. Steady state spectroscopy, atomic force microscopy, electron microscopy and dynamic light scattering methods were used. It was explored how bovine serum albumin affects stability and spectral properties of quantum dots in aqueous media. CdTe–TGA quantum dots in aqueous solution appeared to be not stable and precipitated. Interaction with bovine serum albumin significantly enhanced stability and photoluminescence quantum yield of quantum dots and prevented quantum dots from aggregating. PMID:27502633

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

Aggregation-induced emission enhancement of anthracene-derived Schiff base compounds and their application as a sensor for bovine serum albumin and optical cell imaging.

PubMed

Densil, Simon; Chang, Chien-Huei; Chen, Chia-Ling; Mathavan, Alagarsamy; Ramdass, Arumugam; Sathish, Veerasamy; Thanasekaran, Pounraj; Li, Wen-Shan; Rajagopal, Seenivasan

2018-06-01

Three anthracene-based Schiff base complexes, R1-R3 (R1 = (E)-N´-((anthracen-10-yl)methylene)benzohydrazide; R2 = (E)-1-((anthracen-10-yl)methylene)-4-phenylsemicarbazide; and R3 = (E)-1-((anthracen-10-yl)methylene)-4-phenylthiosemicarbazide) were synthesized from 9-anthracenecarboxaldehyde, benzohydrazide, 4-phenylsemicarbazide and 4-phenylthiosemi-carbazide respectively, and characterized by various spectral techniques. The absorption spectral characteristics of R1-R3 were bathochromically tuned to the visible region by extending the π conjugation. These target compounds were weakly fluorescent in tetrahydrofuran (THF) solution because of rapid isomerization of the C=N double bond in the excited state. However, the aqueous dispersion of R1-R3 in the THF/water mixture by the gradual addition of water up to 90% resulted in an increase in the fluorescence intensity mainly due to aggregation-induced emission enhancement (AIEE) properties. The formation of nanoaggregates of R1-R3 were confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. The compounds R1-R3 are ideal probes for the fluorescence sensing of bovine serum albumin (BSA) and breast cancer cells by optical cell imaging. Copyright © 2018 John Wiley & Sons, Ltd.

Advancing multiscale structural mapping of the brain through fluorescence imaging and analysis across length scales

PubMed Central

Hogstrom, L. J.; Guo, S. M.; Murugadoss, K.; Bathe, M.

2016-01-01

Brain function emerges from hierarchical neuronal structure that spans orders of magnitude in length scale, from the nanometre-scale organization of synaptic proteins to the macroscopic wiring of neuronal circuits. Because the synaptic electrochemical signal transmission that drives brain function ultimately relies on the organization of neuronal circuits, understanding brain function requires an understanding of the principles that determine hierarchical neuronal structure in living or intact organisms. Recent advances in fluorescence imaging now enable quantitative characterization of neuronal structure across length scales, ranging from single-molecule localization using super-resolution imaging to whole-brain imaging using light-sheet microscopy on cleared samples. These tools, together with correlative electron microscopy and magnetic resonance imaging at the nanoscopic and macroscopic scales, respectively, now facilitate our ability to probe brain structure across its full range of length scales with cellular and molecular specificity. As these imaging datasets become increasingly accessible to researchers, novel statistical and computational frameworks will play an increasing role in efforts to relate hierarchical brain structure to its function. In this perspective, we discuss several prominent experimental advances that are ushering in a new era of quantitative fluorescence-based imaging in neuroscience along with novel computational and statistical strategies that are helping to distil our understanding of complex brain structure. PMID:26855758

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.

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

PubMed

Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Shankar, Esaki M; Wong, Kum Thong

2017-01-01

During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

PubMed Central

Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Wong, Kum Thong

2017-01-01

Background During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. Methods We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. Results TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. Conclusion B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection. PMID:28045926

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.

Synthesis and spectral properties of preorganized BODIPYs in solutions and Langmuir-Schaefer films

NASA Astrophysics Data System (ADS)

Marfin, Yuriy S.; Usoltsev, Sergey D.; Kazak, Alexandr V.; Smirnova, Antonina I.; Rumyantsev, Evgeniy V.; Molchanov, Evgeniy E.; Kuznetsov, Vladimir V.; Chumakov, Alexey S.; Glukhovskoy, Evgeny G.

2017-12-01

In order to investigate the influence of molecular structure peculiarities of boron-dipyrrine dyes (BODIPYs) on their properties in solutions and supramolecular organization in Langmuir-Schaefer (LS) films, four new BODIPY dyes with various aliphatic, aromatic or mixed nature meso-subtituents were synthesized and investigated. Spectral characteristics (electronic absorption and fluorescence) of the synthesized compounds in organic solvents and LS-films were studied. Floating monolayers of the BODIPYs were formed from chloroform solutions placed onto water subphase in Langmuir-Blodgett through. Thin films were prepared using the Langmuir-Schaefer technique by the transfer of floating monolayers onto standard polished glass, ITO covered glass or pure silicon substrate. The variation of the dye structure we consider as a preorganization aiming to influence the structure of LS-films. The morphology and structure of the LS-films was examined by fluorescent microscopy, scanning electron microscopy, atomic force microscopy and small angle X-ray diffraction analysis. It was found that the introduced substituents have no substantial influence on the position of the absorption and fluorescence bands in dilute solutions. In contrast, the fluorescent characteristics of the LS-films significantly depend on the substituent nature. Therefore, this strategy could be used for the direct tuning of compounds fluorescent properties in LS-films. Concerning the LS-film surface characteristics it was proved that the films are homogeneous, without disruptions and only some widely-spaced microcrystals could be observed. With respect to the LS-film structure, the change of the substituents introduced to the BODIPY molecule did not influence the average given periodicity of layers (d = 0.3-0.4 nm). This value corresponds to a single-layer arrangement of BODIPY molecules located parallel to the substrate surface. Nevertheless, the diffraction peak intensities depended on the molecular structure of BODIPYs and therefore the structurization in thin films. Moreover, the combination of the rigid phenyl moiety with long alkyl chains in one compound completely suppresses the aggregation of molecules maintaining the intense fluorescence in thin films. On the basis of used range of experimental and calculation methods the intralayer and interlayer structures were proposed. Intermolecular hydrogen bond formation and π-π staking of the BODIPY cores were found to be the structure forming forces during the films manufacturing, resulting the differences in crystallinity of the materials. While the alkyl-substituents prevent the type of interactions and suppress the association of the dyes and formation of excimers. Compounds under investigation show a manifestation of the intense solvatochromic properties which allow their application as sensors, including naked eye sensorics for solution polarity. Besides, the obtained results broaden prospective of functional materials usage based on BODIPY thin films as components of optoelectronics.