Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation

Imaging of gap junction proteins, the connexins, has been performed in tissue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent proteins such as Green Fluorescent Protein. These two approaches have been used to gain information about protein localization or trafficking at light microscopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap junction proteins in whole cells at the electron microscopic level has been difficult to achieve because of the fixation sensitivity of most gap junction antibodies. To obtain reasonable sensitivity, immunoperoxidase procedures are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniques and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using correlative microscopy, we can not only localize connexin pools or structures, but also discover what other cellular substructures interact with gap junction proteins. The use of tyramide signal amplification techniques is necessary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescence photooxidation technique provides a high-resolution method for staining of proteins in cells. Unlike colloidal gold-based methods, fluorescence photooxidation allows for three-dimensional localization using high-voltage electron microscopy.     

Quantitative aspects of synapses on Golgi-impregnated neurons.

With the classical Golgi techniques, numerous types of neurons can be distinguished in the cerebral cortex, each with a specific dendritic geometry and pattern of axonal ramifications. In the present review we describe two techniques which allow quantification of synapses on identified neurons: (1) Golgi-rapid impregnation-gold toning-electron microscopy, and (2) Golgi-Kopsch impregnation-gold toning-electron microscopy in combination with staining of the tissue with ethanolic phosphotungstic acid (E-PTA). Both techniques were applied on neurons in the visual cortex of young and adult rabbits. By means of rotating and tilting specimens in the electron microscope, the nondistinctive ultrastructure of obliquely sectioned synapses can be circumvented, leading to precise estimates of asymmetrical vs. symmetrical synapses without complete reconstruction of the neuron.     

Application of the Golgi/electron microscopy technique for cell identification in immunocytochemical, retrograde labeling, and developmental studies of hippocampal neurons.

In this study the Golgi/electron microscopy (EM) technique has been used for an analysis of the fine structure, specific synaptic connections, and differentiation of neurons in the hippocampus and fascia dentata of rodents. In a first series of experiments the specific synaptic contacts formed between cholinergic terminals and identified hippocampal neurons were studied. By means of a variant of the section Golgi impregnation procedure, Vibratome sections immunostained for choline acetyltransferase, the acetylcholine-synthesizing enzyme, were Golgi-impregnated in order to identify the target neurons of cholinergic terminals in the hippocampus. It could be shown with this combined approach that cholinergic septohippocampal fibers form a variety of synapses with different target structures of the Golgi-impregnated and gold-toned hippocampal neurons. In this report cholinergic synapses on the heads of small spines, the necks of large complex spines, dendritic shafts, and cell bodies of identified dentate granule cells are described. The variety of cholinergic synapses suggests that cholinergic transmission in the fascia dentata is a complex event. Next, the Golgi/EM technique was applied to Vibratome sections that contained retrogradely labeled neurons in the hilar region of the fascia dentata following horseradish peroxidase (HRP) injection into the contralateral hippocampus. With this combined approach some of the hilar cells projecting to the contralateral side were identified as mossy cells by the presence of retrogradely transported HRP in thin sections through these Golgi-impregnated and gold-toned neurons. Our findings suggest that the mossy cells are part of the commissural/associational system terminating in the inner molecular layer of the fascia dentata. They are mainly driven by hilar collaterals of granule cell axons that form giant synapses on their dendrites. Finally, the Golgi/EM procedure was used to study the differentiation and developmental plasticity of hippocampal and dentate neurons in transplants and slice cultures of hippocampus. Under both experimental conditions, the differentiating neurons are deprived of their normal laminated afferent innervation but develop their major cell-specific characteristics including a large number of postsynaptic structures (spines). As revealed in thin sections of gold-toned identified cells, all these spines formed synapses with presynaptic boutons suggesting sprouting of the transplanted and cultured neurons, respectively. Altogether, the present report demonstrates the usefulness of the Golgi/EM technique, particularly of the section impregnation procedure, for a variety of studies requiring the identification of individual neurons at the ultrastructural level.     

The beauty of the yeast: live cell microscopy at the limits of optical resolution

The yeast Saccharomyces cerevisiae is a very powerful system for cell biological research. Recent advances in electronic light microscopy together with the application of green fluorescent protein and other in vivo staining techniques have allowed novel and exciting insights into structural organization and dynamics of cells as small as yeast. Methods for staining yeast for microscopic inspection and for introducing tags for localization studies of proteins in living or fixed cells are summarized. Electronic light microscopy, video/deconvolution methods, and confocal laser scanning microscopy as novel tools for structural analyses, and their practical applications in yeast, are discussed.     

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.     

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.     

Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison

Despite the numerous studies performed in an attempt to clarify the issue, the mechanism of action of salivary histatins remains unclear. The aim of the present study was to correlate histatin-induced morphological changes in Candida albicans by fluorescence microscopy (FM), transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM). Each of the fluorescent dyes used by FM (i.e., tetramethylrhodamine methyl ester perchlorate for mitochondrial potential, Lysotracker for lysosome acidic compartment, and 4',6-diamino-2-phenylindole dihydrochloride for DNA) exhibited a specific staining in control cells. Following histatin treatment, we observed a recurring staining pattern, corresponding to fluorescence concentration along the cell periphery, suggesting a loss of dye specificity. To assess histatin-induced cytoplasmic modifications, ultrastructural analysis was then carried out. After treatments with histatins, TEM revealed characteristic intracellular modifications including: vacuole overgrowth, nuclear disappearance, loss of organelle identity, as well as the appearance of electron-dense membranes, likely of mitochondrial origin. Additionally, structures resembling autophagosomes were occasionally observed. By HRSEM, mitochondrial swelling was invariably the first sign of a histatin-induced effect. Other modifications included intracellular membrane disarrangement, organelles in disarray, and a large central cavity with deformed bodies displaced to the cell periphery, similar to what was detected by TEM. In summary, our study illustrates the occurrence of ultrastructural modifications following administration of histatins. Observations made with FM, TEM, and HRSEM provided different views of the same signs, demonstrating a definite action of histatins on C. albicans morphology. The possible functional meanings of these morphological results is discussed in light of the most recent biochemical data on histatin fungicidal activity.     

A novel approach for correlative light electron microscopy analysis

Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three‐dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Cautionary observations on preparing and interpreting brain images using molecular biology-based staining techniques

Though molecular biology-based visualization techniques such as antibody staining, in situ hybridization, and induction of reporter gene expression have become routine procedures for analyzing the structures of the brain, precautions to prevent misinterpretation have not always been taken when preparing and interpreting images. For example, sigmoidal development of the chemical processes in staining might exaggerate the specificity of a label. Or, adjustment of exposure for bright fluorescent signals might result in overlooking weak signals. Furthermore, documentation of a staining pattern is affected easily by recognized organized features in the image while other parts interpreted as "disorganized" may be ignored or discounted. Also, a higher intensity of a label per cell can often be confused with a higher percentage of labeled cells among a population. The quality, and hence interpretability, of the three-dimensional reconstruction with confocal microscopy can be affected by the attenuation of fluorescence during the scan, the refraction between the immersion and mounting media, and the choice of the reconstruction algorithm. Additionally, visualization of neurons with the induced expression of reporter genes can suffer because of the low specificity and low ubiquity of the expression drivers. The morphology and even the number of labeled cells can differ considerably depending on the reporters and antibodies used for detection. These aspects might affect the reliability of the experiments that involves induced expression of effector genes to perturb cellular functions. Examples of these potential pitfalls are discussed here using staining of Drosophila brain.     

Electron microscopy of cultured angiogenic endothelial cells

Angiogenesis is a multi-step process involving migration, proliferation, and a specific spatial arrangement of endothelial cells. On the basis of a model of cultured microvascular endothelial cells derived from the bovine corpus luteum, all stages of in vitro angiogenesis as well as intussusceptive remodeling were characterized by scanning and transmission electron microscopy. To preserve the delicate three-dimensional cellular structures for electron microscopy, modified processing techniques for both transmission and scanning electron microscopy including micro-corrosion casting of cultured cells were established. The detailed results on morphological alterations and cellular interactions confirmed and complemented earlier studies of in vitro angiogenesis. Electron microscopy proved to be an efficient tool for detection and supervision of all major endothelial differentiation processes resembling in vivo conditions that are generally considered important in a realistic in vitro model of angiogenesis: occurrence of function-related cellular junctions; development of specific surface features indicating cellular polarity; production of extracellular matrix material; mechanisms leading to the formation of an internal lumen; specific spatial arrangement of endothelial cells within capillary-like networks; detachment of apoptotic cells as well as intussusception of specific cells within the course of vascular remodeling. The abundance of quickly available information provided by electron microscopic approaches may be useful for subsequent, e.g., biochemical or molecular, studies and thus delivers important controls for further experimental designs.     

Immunohistochemistry of GluR1 subunits of AMPA receptors of rat cerebellar nerve cells

The localization of GluR1 subunits of ionotropic glutamate receptors in the glial cells and inhibitory neurons of cerebellar cortex and their association with the climbing and parallel fibers, and basket cell axons were studied. Samples of P14 and P21 rat cerebellar cortex were exposed to a specific antibody against GluR1 subunit(s) of AMPA receptors and were examined with confocal laser scanning microscopy. GluR1 strong immunoreactivity was confined to Purkinje cell and the molecular layer. Weak GluR1 immunoreactivity was observed surrounding some Golgi cells in the granule cell layer. Intense GluR1 immunoreactivity was localized around Purkinje, basket, and stellate cells. Purkinje cells expressed strong GluR1 immunoreactivity surrounding the cell body, primary dendritic trunk and secondary and tertiary spiny dendritic branches. Marked immunofluorescent staining was also detected in the Bergmann glial fibers at the level of middle and outer third molecular layer. Positive immunofluorescence staining was also observed surrounding basket and stellate cells, and in the capillary wall. These findings suggest the specific localization of GluR1 subunits of AMPA receptors in Bergmann glial cells, inhibitory cerebellar neurons, and the associated excitatory glutamatergic circuits formed by climbing and parallel fibers, and by the inhibitory basket cell axons     

Saccharomyces cerevisiae samples stained with FUN-1 dye can be stored at −20°C for later observation

FUN‐1, a fluorescent vital dye, has been observed to form cylindrical intravacuolar structures within the vacuoles of metabolically active yeast cells. FUN‐1 staining, which begins as a diffuse pool of fluorescent cytoplasmic stain, uses an unknown endogenous biochemical processing mechanism to compact and form orange‐red cylindrical intravacuolar structures within the cell vacuole. In the clinical setting, FUN‐1 is primarily used for identification of fungal infection. FUN‐1 is utilized in the laboratory to distinguish between metabolically active and dead fungal cells. Although this stain is useful for distinguishing between live and dead fungal dead cells, few studies have utilized this chemical. This lack of use in the scientific community may be due to the requirement that cells are visualized directly after staining. Thus, it would be of interest to be able to stain cells and store them for later use. Our lab examined the longevity of cylindrical intravacuolar structures in two strains of Saccharomyces cerevisiae stained with FUN‐1 and stored at −20°C. We found that cylindrical intravacuolar structures could be reliably observed and imaged utilizing differential interference contrast microscopy and fluorescence microscopy for 21 days. We also observed that cells stained with FUN‐1 would resume propagation on yeast extract, peptone, dextrose (YPD) plates after being frozen at −20°C for 21 days. These modifications to the published procedure for FUN‐1 dye staining should allow for a more prevalent and less time sensitive use of this important biological tool.     

Morphological changes in mouse embryos cryopreserved by different techniques

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3-3'-diaminobenzidine and immunocytochemistry.

We describe the application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver. For automatic detection by light and electron microscopy peroxisomes must be stained with the alkaline DAB procedure for catalase. There is a good agreement between the results obtained by conventional morphometric techniques and by automatic image analysis of DAB-stained electron microscopic preparations. Moreover, the image analyzer may be used in conjunction with a light microscope for evaluation of semithin sections (1-0.25 microns), provided the section thickness factor is taken into consideration. This latter approach has proven highly efficient in estimation of peroxisome proliferation. The limitations of this method and the relevance of volume density as a reliable morphometric parameter for evaluation of peroxisome proliferation are discussed. In the second part of this study we present the application of image analysis for quantitation of alterations of individual peroxisomal enzyme proteins after treatment with bezafibrate in immunogold stained ultrathin sections. There is good agreement between the results of quantitative immunocytochemistry and Western (immuno) blot analysis of highly purified peroxisomal fractions. In our experience quantitative immunoelectron microscopy provides a versatile, highly sensitive, and efficient method for detection of modulations of various proteins in peroxisomes. Finally the limitations and prospects of quantitative immunocytochemistry for investigation of peroxisomal proteins are discussed.     

Six-color segmentation of multicolor images in the infection studies of Listeria monocytogenes

Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis.     

Intracellular distribution of actin in cells of the orgen of Corti: A structural basis for cell shape and motility

Immunofluorescence staining and phalloidin labeling have provided localization of actin in the sensory and supporting cells of the inner ear at the light microscopic level. However, with electron microscopy, neither actin nor actin filaments have been found in the outer hair cell body. This paper describes various techniques utilized to preserve and identify cytoplasmic actin at the ultrastructural level. Post-embedding staining of Lowicryl K4M sections, pre-embedding staining of permeabilized cells of the organ of Corti, pre-embedding staining of vibratome sections, and pre-embedding staining of permeabilized dissociated cells documented the presence of actin, but each of these techniques was best suited to localize actin in specific parts of the cell. Cytoplasmic actin was labeled when isolated cells were lightly fixed and membranes were permeabilized with detergent—conditions under which the cell ultrastructure was compromised. Under conditions of optimal fixation, cytoplasmic filaments embedded in the dense granular matrix of the hair cell cytoplasm were observed.     

Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV-1 on the surface of HIV-1-infected H9-lymphocytes

Immunofluorescence staining techniques at present, when applied to follow the expression of HIV-1-specific antigens on infected cells, only give the information that the antigens detected are localized in the outer region of the membrane of the infected cell. We therefore set up a procedure using magnetic polystyrol particles coated with antibodies specific for the HIV-1 antigens under study, in combination with scanning electron microscopy. We were able to demonstrate that p24 and p18 structural antigens are clearly expressed on the surface of HIV-1-infected H9 lymphocytes. This means that there was no steric hindrance for structures of cell-like size specific for HIV-1 antigens to interact with their target antigens. Other antigens may be hidden in membrane structures and are therefore inaccessible, for example, to the beads used here, which were of a similar size to antigen-specific cells in vivo. The results of this model system must be seen with respect to the interaction of antigen-specific cell-mediated immunity with full antibody-dependent cellular cytotoxicity, or without cytotoxic T lymphocytes, the mediator function of antibodies.     

Autofluorescence removal using a customized filter set

Quantitative fluorescence microscopy is severely hindered by intrinsic autofluorescence (AF). Endogenous fluorescent molecules in tissue and cell samples emit fluorescence that often dominates signals from specific dyes. This makes AF removal critical to the development and practice of quantitative fluorescence microscopy. In this study, we showed that AF signal could be separated from specific signal using a customized filter set. The filter set used the same excitation and beam splitter as the standard filter set, but the emission filter was red‐shifted 40–60 nm from the peak of the specific dye. This filter set configuration collected mostly AF with minimum contribution from the specific dye. A linear transformation of AF images was required to correct for the difference in exposure and filter configuration. The constants (slope and intercept) in linear transformation were obtained through a pixel to pixel comparison between AF images (no staining) obtained by the standard filter set and the customized AF filter set. After staining of specific dye, the standard filter collecting target dye spectra was used to capture both target signal and AF, whereas customized filter was used to capture only AF. AF removal was accomplished by subtracting the linear transformed AF image from the image obtained from the standard filter. To validate our approach, we examined weak staining of androgen receptor in an AF abundant prostate tissue sample. Our method revealed a similar but cleaner nuclear staining of androgen receptor in a specimen, when compared to a traditional autofluorescence removal method. Microsc. Res. Tech., 76:1007–1015, 2013. © 2013 Wiley Periodicals, Inc.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.