Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Dynamic confocal scanning laser microscopy methods for studying milk protein gelation and cheese melting

Dynamic confocal scanning laser microscopy (CSLM) methods were developed to enable observation of milk protein gelation and cheese melting. Protein aggregation and the formation of gel networks in renneted full-fat and low-fat milks and glucono-δ-lactone (GDL)-acidified skim milks were observed by CSLM and observations correlated with increases in shear modulus (G′) and dynamic viscosity (η*) as determined by dynamic amplitude oscillatory rheology. Confocal scanning laser microscopy observation of low-fat and full-fat cheeses showed changes in fat distribution and an increase in staining intensity during cheese melting.     

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

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.     

High resolution SEM characterization of nano‐precipitates in ODS steels

The performance of the present‐day scanning electron microscopy (SEM) extends far beyond delivering electronic images of the surface topography. Oxide dispersion strengthened (ODS) steel is on of the most promising materials for the future nuclear fusion reactor because of its good radiation resistance, and higher operation temperature up to 750°C. The microstructure of ODS should not exceed tens of nm, therefore there is a strong need in a fast and reliable technique for their characterization. In this work, the results of low‐kV SEM characterization of nanoprecipitates formed in the ODS matrix are presented. Application of highly sensitive photo‐diode BSE detector in SEM imaging allowed for the registration of single nm‐sized precipitates in the vicinity of the ODS alloys. The composition of the precipitates has been confirmed by TEM‐EDS.     

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy

Thin cross-sections of human hairs were investigated by scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross-sections of hairs.     

A structural study of the bovine vaginal fluid at estrus.

The present study describes the structural components of the bovine vaginal fluid at estrus by scanning electron microscopy (SEM) following critical point- and freeze-drying preparation procedures. Confocal scanning laser microscopy (CLSM) was also used to evaluate the structural integrity of samples, and a control sample was assessed by adding sperm to the vaginal fluid. Samples were collected from 10 cows at the time of artificial insemination, prepared for SEM by using critical point- and freeze-drying procedures, gold coated, and observed by SEM. Mesh size and filament thickness were measured with an image analyzer. Of the 10 samples processed, 4 were considered altered following critical point drying. Compaction and lack of filaments were observed in these samples. A small area of one sample showed a honey comb-like structure when freeze drying was used. Nonoriented filaments with different thicknesses and with a network-like structure were observed throughout the remainder of the samples. Filaments throughout all samples were also observed by CSLM. After critical point drying, the mesh area ranged from 0.8 to 101.4 microns 2; the minor axis from 0.7 to 10.8 microns; and filament thickness from 40 to 442 nm. Using freeze drying, the mesh area ranged from 0.9 to 493.8 microns 2; the minor axis from 0.7 to 27.5 microns; and filament thickness from 40 to 800 nm. When samples were freeze dried, mesh values were similar to the interstrand channels observed by CSLM. In sperm-vaginal fluid samples, following critical point- or freeze-drying procedures, spermatozoa were oriented randomly in the vaginal fluid and did not seem to alter filamentous structure. Our data suggest that the freeze-drying procedure better preserves the true structural dimensions of the vaginal fluid. Furthermore, the filamentous structure of the vaginal fluid does not appear to impede sperm transport.     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.     

Radon track imaging in CR-39 plastic detectors using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) has been used to provide the first images of radon track populations in two external CR-39 plastic detectors. Measurements of variables including track area distribution and estimates of the angle of track inclination (dip) derived from surface CSLM sections are presented. CSLM depth slices, combined with three-dimensional (3D) visualization techniques, provide a new, non-destructive way of examining the 2D and 3D geometry of the etched tracks within solid-state nuclear track detectors that may prove useful in complementing existing optical microscopy methods.     

Microfractography of granitic rocks under confocal scanning laser microscopy

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three-dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures, and the rock-forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.     

Determination of the water droplet size distribution of fat spreads using confocal scanning laser microscopy

Knowledge of the water droplet size distribution of fat spreads is necessary for the development and production of high quality microbiological safe products. Fat spreads are water‐in‐oil emulsions. The water droplet size distribution can be determined by confocal scanning laser microscopy (CSLM) after staining the fat with Nile Red. The profiles of the non‐fluorescent water droplets in the 2D images are identified and measured using image analysis. The ‘true’ water droplet size distribution is calculated from the distribution of the measured profile diameters using a Wicksell transformation of log‐normal distributions. The influence of the fluorescent staining and CSLM parameters on the information were studied. The CSLM method was tested on fat spreads with a fat content ranging from 40% to 80%. The results were compared with those obtained by nuclear magnetic resonance spectroscopy (NMR). The distribution parameters [volume weighed geometric mean diameter (D?_3,3) and the standard deviation (σ) of the logarithm of the droplet diameter] calculated for 80% fat spreads are in good agreement with those obtained by NMR (within ± 7% relative). Small differences were found for 65% fat spreads and large differences were identified for 40% fat spreads. The precision for the determination of the D?_3,3 value by CSLM is worse than that of NMR, even when three images were used to calculate this parameter [3–11% and 1–6% relative standard deviation (RSD), respectively]. The precision for the determination of exp(σ) by CSLM is comparable or better than that of NMR (1–5% and 3–6% RSD, respectively). CSLM proved to be a reliable method for the determination of the water droplet size distribution of margarines (80% fat). The advantage of CSLM compared to NMR is that visual information is given about the water droplet size distribution in the sample.     

In situ analysis of microbial consortia in activated sludge using fluorescently labelled,rRNA-targeted oligonucleotide probes and confocal scanning laser microscopy

Activated sludge flocs are complex consortia of various micro-organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.     

A comparison of imaging methodologies for 3D tissue engineering

Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current "gold standard" for imaging three-dimensional tissue engineered constructs, histology.     

激光扫描共聚焦显微镜技术的发展及应用

激光扫描共聚焦显微术是先进的分子和细胞生物学研究技术。它在荧光显微镜成像的基础上加装激光扫描装置,结合数据化图像处理技术,采集组织和细胞内荧光标记图像。在亚细胞水平观察钙等离子水平的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。由于它的应用范围较广泛,已成为形态学、分子细胞生物学、神经科学和药理学等研究领域中很重要的研究技术。     

Ultrastructural investigation of neurons identified and localized using the confocal scanning laser microscope

Studies of labeled neurons at the light-microscopic level often pinpoint a substructure of particular interest, i.e., a synapse or a spine. An ultrastructural investigation would explain a lot about how these structures arose, how they function, and how they are regulated. Finding a small region in a large block can require constant checking during sectioning, until past the structure. In our pursuit of the synaptic structure of varicosities on the axons of neurons identified physiologically and morphologically at the light level, we have combined confocal scanning laser microscopy (CSLM) with conventional and high-voltage electron microscopy (EM). CSLM images were collected in the reflection mode to view neurons filled with horseradish peroxidase and stained with nickel-intensified diaminobenzidine, which is compatible with EM. The CSLM optical sections provided a record of what one should expect to see at regular intervals throughout the depth of the tissue block. We have shown that the CSLM greatly simplified the task of localizing small structures in brain tissue prepared for EM.     

Monte Carlo simulation of secondary electron and backscattered electron images in scanning electron microscopy for specimen with complex geometric structure

A new Monte Carlo technique for the simulation of secondary electron (SE) and backscattered electron (BSE) of scanning electron microscopy (SEM) images for an inhomogeneous specimen with a complex geometric structure has been developed. The simulation is based on structure construction modeling with simple geometric structures, as well as on the ray-tracing technique for correction of electron flight-step-length sampling when an electron trajectory crosses the interface of the inhomogeneous structures. This correction is important for the simulation of nanoscale structures of a size comparable with or even less than the electron scattering mean free paths. The physical model for electron transport in solids combines the use of the Mott cross section for electron elastic scattering and a dielectric function approach for electron inelastic scattering, and the cascade SE production is also included.     

Multiphoton fluorescent images with a spatially varying background signal: a ML deconvolution method

By means of multiphoton laser scanning microscopy, neuroscientists can look inside the brain deeper than has ever been possible before. Multiphoton fluorescent images, as all optical images, suffer from degradation caused by a variety of sources (e.g. light dispersion and absorption in the tissue, laser fluctuations, spurious photodetection and staining deficiency). From a modelling perspective, such degradations can be considered the sum of stochastic noise and a background signal. Among the methods proposed in the literature to perform image deconvolution in either confocal or multiphoton fluorescent microscopy, Vicidomini et al. (2009) were the first to incorporate models for noise (a Poisson process) and background signal (spatially constant) in the context of regularized inverse problems. Unfortunately, the so-called split-gradient deconvolution method (SGM) they used did not consider possible spatial variations in the background signal. In this paper, we extend the SGM by adding a maximum-likelihood estimation step for the determination of a spatially varying background signal. We demonstrate that the assumption of a constant background is not always valid in multiphoton laser microscopy and by using synthetic and actual multiphoton fluorescent images, we evaluate the face of validity of the proposed method, and compare its accuracy with the previously introduced SGM algorithm.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.     

Three-Dimensional imaging of fertilization and early development

The field of biological microscopy has recently enjoyed major technical advances, exemplified by the development of field-emission low-voltage scanning electron microscopes and laser scanning confocal light microscopes. In addition, computer processing of microscopical data is revolutionizing the way morphological information is imaged. In this paper, we illustrate methods by which this new technology can be used to examine events in fertilization and early development in three dimensions. Different types of specimen preparation protocols, using both echinoderm and mammalian gametes and embryos, are evaluated for their ability to preserve accurately the threedimensional organization of these specimens for imaging by both low-voltage scanning electron microscopy and laser scanning confocal light microscopy.