Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation

Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.     

Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy

The addition of the alcohol iso‐butanol (2‐methylpropan‐1‐ol) to water was found to improve the post‐staining procedures for semi‐thin and ultrathin resin sections, for both light and electron microscopy. Stain penetration was enhanced with samples embedded in both acrylic and epoxy resins and provided structural information not previously seen. These improvements were found with general (non‐specific) stains and a fluorescence stain for light microscopy, as well as for a range of heavy metal stains for electron microscopy. The use of this water/solvent medium also gave improved results when used in a variety of immunogold labelling procedures, resulting in a greater specific label density without affecting background gold levels. The use of this solvent/water medium may have wider applications for other types of staining.     

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.     

Comparative evaluation of vegetable matter involved lesions with oral parasitic infections in the oral cavity

The current research aims to perform a comparative evaluation of vegetable matter involved lesions with oral parasitic infections found in oral mucosa, presenting histochemical methods to differentiate their microscopic similarities. Eight cases were selected out of a sample of 1.975 reports from a single Oral and Maxillofacial Pathology Service of the author's institution from 2012 to 2019. Specimens were examined by hematoxylin and eosin staining (HE), periodic acid-Schiff (PAS) staining, Gomori–Grocott staining, Ziehl–Neelsen staining, Giemsa, and mucicarmine staining. Microscopic analysis included fluorescence, polarized light, and confocal microscopy. Microscopically, in HE coloration, hookworm eggs showed as eosinophilic. Inflammatory multinucleated giant cells and lymphocytes, were usually related to the nematode eggs, forming an intense inflammatory infiltrate. Biofluorescent properties of eggs and larvae revealed to be sensitive in the detection of parasitic structures contrasting with the inflamed connective tissue. Vegetable presence was confirmed by polarized light microscopy and it was found to be associated with microbial biofilms. Confocal microscopy has showed to be an excellent method for morphotype differentiation of parasitic eggs. Parasitic infection and vegetable matter displayed similarities in the inflammatory response, but the latter can rot and agglomerate biofilms. The microscopic diagnosis of such infections requires the interpretation of challenging morphological features since the parasites are usually sectioned and mixed with an inflammatory reaction. These histochemical approaches proved to be excellent to distinguish both lesions.     

Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described.     

Observations on the nucleolar staining by ethanolic phosphotungstic acid.

In addition to the already known reactivity of heterochromatin masses and synaptonemal complexes for ethanolic phosphotungstic acid, nucleoli from Sertoli cells show a preferential electron microscopic staining of the pars fibrosa. This ultrastructural pattern can be correlated with intranucleolar differentiations observed in light microscopy after staining of semithin sections with Unna's polychrome blue.     

Attachment and entry of Candida famata in monocytes and epithelial cells

Candida albicans is considered the main pathogenic yeast responsible for a multitude of infective disorders. However, other yeasts, such as Candida famata, are being recognized as potential emerging pathogens that cause several types of infections in humans and animals. Consequently, we have investigated the adhesion and internalization of Candida famata into monocytes and epithelial cells. The interaction of the yeast with the cells is very rapid and takes place during the first 15 min of injection. However, the affinity of the yeast for the cells varies, THP-1 (human monocytes) being the highest and followed in decreasing order by HeLa (human carcinoma), HaCaT, and Pam-212 (human and mouse keratinocytes, respectively). Heat inactivation or treatment with nystatin, significantly decreases yeast adhesion to cells. Immunofluorescence, as well as scanning and transmission electron microscopy, reveals that cell lines are able to internalize C. famata. At 48 h after infection, most of the yeasts located inside cells appear degraded, but some yeasts recovered from lysed cells, were still viable. Adhesion and internalization of C. famata into HeLa cells were found to be lower than those of C. albicans and C. glabrata, but higher than those of S. cerevisiae. In addition, infection with C. famata results in actin microfilaments rearrangement. This article presents novel data in the interaction of this pathogenic yeast with mammalian cells.     

Intercellular junctions in FANFT-induced carcinomas of rat urinary bladder in tissue culture: in situ thin-section, freeze-fracture, and scanning electron microscopy studies.

This paper describes a set of simple methods for comparative light and electron microscopy studies on tissue cultured tumour cells derived from both noninvasive and invasive carcinogen-induced rat urinary bladder carcinomas. Cells are grown on Thermanox plastic coverslips and fixed in situ. Each plastic coverslip is then divided with scissors into four parts: the first is processed for light microscopy, the second for thin-section electron microscopy, the third for freeze-fracture electron microscopy, and the fourth for scanning electron microscopy. In some experiments, portions of the culture which have first been examined by light microscopy are subsequently prepared for electron microscopy. In this way, the culture conditions are kept constant and comparison of structural features (i.e. intercellular junctions) by several preparative techniques is possible. Noninvasive and invasive rat bladder tumour cells, characterized by numerous pleomorphic microvilli, have normal zonulae occludentes at the apices of lateral surfaces of tumour cells in all cultures. In some areas of invasive tumour cells, occludens junctions are focally attenuated, consisting of only one or two strands, and occasionally the strands are discontinuous. Gap junctions, type PF-1, as well as numerous demosomes are present in all cell lines. Thus, intercellular junctions in noninvasive and invasive rate bladder epithelial cell lines bear a striking resemblance to those previously described in the comparable solid primary tumours. These culture systems may be useful for studying factors which influence the formation of intercellular junctions during malignant transformation.     

Pushing the resolution limits in cryo electron tomography of biological structures

Cryo electron tomography is a three-dimensional imaging technique that is suitable for imaging snapshots of the structural arrangements of biomolecular complexes and macromolecules, both in vitro and in the context of the cell. In terms of attainable resolution, cryo electron tomographic reconstructions now show resolvable details in the 5-10 nm range, connecting optical microscopy with molecular imaging techniques. In view of the current developments in super-resolution light microscopy and correlative light and electron microscopy, cryo electron tomography will be increasingly important in structural biology as a tool to bridge light microscopy with molecular imaging techniques like NMR, X-ray diffraction and single particle electron microscopy. In cell biology, one goal, often referred to as visual proteomics, is the molecular mapping of whole cells. To achieve this goal and link cryo electron tomography to these high-resolution techniques, increasing the attainable resolution to 2-5 nm is vital. Here, we provide an overview of technical factors that limit the resolution in cryo electron tomography and discuss how during data acquisition and image processing these can be optimized to attain the highest possible resolution. Also, existing resolution measurement approaches and current technological developments that potentially increase the resolution in cryo electron tomography are discussed.     

Transmission X-ray microscopy of intact hydrated PtK2 cells during the cell cycle

Transmission X-ray microscopy makes it possible to investigate biological specimens, i.e. cells and cell organelles, in their natural wet environment. The main processes determining the contrast in X-ray microscopy are photoelectric absorption and phase shift. X-ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X-ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X-ray microscopy in biomedical research are discussed.     

Methodical investigations for quantitative image analysis on cells of the female genital tract (author's transl)]

An earlier published [8] image analysis system for cytology is tested on gynecological material. At first the question for the staining method which is optimal for scanning-photometrical measurements is investigated. In addition the wavelength of the light which reproduces best the nuclear structure must be found. Feulgen-stained preparations scanned at 570nm allow a more secure sorting of superficial cells against intermediary cells than those in the classical Papanicolaou-stain at white light. Discoloring of Papanicolaou-preparations and Feulgen staining afterwards was accomplished without technical problems and there were not detectable any qualitative differences with directly Feulgen-stained preparations by means of image analysis. With this method it is possible to recognize structural changes in nuclei of vaginal epithelium during cell differentation.     

3D visualization of subcellular structures of Schizosaccharomyces pombe by hard X-ray tomography

Cellular structures of the fission yeast, Schizosaccharomyces pombe, were examined by using hard X-ray tomography. Since cells are nearly transparent to hard X-rays, Zernike phase contrast and heavy metal staining were introduced to improve image contrast. Through using such methods, images taken at 8 keV displayed sufficient contrast for observing cellular structures. The cell wall, the intracellular organelles and the entire structural organization of the whole cells were visualized in three-dimensional at a resolution better than 100 nm. Comparison between phase contrast and absorption contrast was also made, indicating the obvious advantage of phase contrast for cellular imaging at this energy. Our results demonstrate that hard X-ray tomography with Zernike phase contrast is suitable for cellular imaging. Its unique abilities make it have potential to become a useful tool for revealing structural information from cells, especially thick eukaryotic cells.     

Involvement of PCH family proteins in cytokinesis and actin distribution

Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.     

Lymphatic networks in the periodontal tissue and dental pulp as revealed by histochemical study.

The structural organization and fine distribution of the lymphatic networks in the periodontal tissues (gingiva, periodontal membrane, and alveolar process) and dental pulp of animals and humans were reviewed with special reference to histochemical examination by light and electron microscopy. The distinction between lymphatics and blood vessels was made on cryostat sections of undecalcified and calcified teeth treated with EDTA solution and whole mount preparations of periodontal membranes using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. This staining procedure allowed lymphatic vessels in the periodontal tissue and dental pulp to be differentiated from blood vessels. The specificity and localization of the enzyme reactions were confirmed by comparative histochemical studies of the same specimen with light microscopy and scanning or transmission electron microscopy. Well-developed 5'-Nase-positive lymphatic networks were observed on the tissue sections and whole mount preparations of the gingiva, periodontium, and dental pulp. More lymphatic vessels were seen in the root area of the periodontium than in the cervical area. In the dental pulp, lymphatic vessels were more numerous in the central part than in the peripheral odontoblastic layer. These distributions of the lymphatic capillary networks are discussed in relation to their ability to supply lymph to the teeth.     

Morphological and functional characterization of circulating hemocytes using microscopy techniques

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi‐granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule‐containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid–Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell‐mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase‐mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme‐agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.     

Microwave-accelerated cytochemical stains for the image analysis and the electron microscopic examination of light microscopy diagnostic slides

Recent studies in our laboratories have shown how microwave (MW) irradiation can accelerate a number of tissue-processing techniques, especially staining, to aid in the preparation of single specimens on glass microscope slides or coverslips for examination by light microscopy (and electron microscopy, if required) for diagnostic purposes. Techniques have been developed, which give permanently stained preparations, that can be studied initially by light microscopy, their areas of interest mapped, and computer-automated image analysis performed to obtain quantitative information. This is readily performed after MW-accelerated staining with silver methenamine by the Giammara-Hanker PATS or PATS-TS reaction. This variation of the PAS reaction gives excellent markers for specific infectious agents such as lipopolysaccharides for gram-negative bacteria or mannans for fungi. It is also an excellent stain for glycogen and basement membranes and an excellent marker for type III collagen or reticulin in the endoneurium or perineurium of peripheral nerve or in the capillary walls. Our improved MW-accelerated Feulgen reaction with silver methenamine for nuclear DNA is useful to show the nuclei of bacteria and fungi as well as of cells they are infecting. Improved coating and penetration of tissue surfaces by thiocarbohydrazide bridging of ruthenium red, applied under MW-acceleration, render biologic specimens sufficiently conductive for SEM so that sputter coating with gold is unnecessary. The specimens treated with these highly visible electron-opaque stains can be screened with the light microscope after mounting in polyethylene glycol(PEG) and the structures or areas selected for EM study are mapped with a Micro-Locator? slide. After removal of the water soluble PEG the specimens are remounted in the usual EM media for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) study of the mapped areas. By comparing duplicate smears from areas of infection, such as two coverslips of buffy coat smears of blood from a patient with septicemia, the microorganisms responsible can occasionally be classified for antimicrobial therapy long before culture results are available; gram-negative bacteria are positive with the Giammara-Hanker PATS-TS stain, and gram-positive bacteria are positive with the SIGMA HT40 Gram stain. The gram-positive as well as gram-negative bacteria are both initially stained by the crystal violet component of the Gram stain. The crystal violet stain is readily removed from the gram-negative (but not the gram-positive) bacteria when the specimens are rinsed with alcohol/acetone. If this rinse step is omitted, the crystal violet remains attached to both gram-negative and gram-positive bacteria. It can then be rendered insoluble, electron-opaque, and conductive by treatment with silver methenamine solution under MW-irradiation. This metallized crystal violet is a more effective silver stain than the PATS-TS stain for a number of gram-negative spirochetes such as Treponema pallidurn, the microbe that causes syphilis     

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.     

Scanning electron and light microscopy of foliar epidermal characters: A tool for plant taxonomists in the identification of grasses

SEM and light microscopy are of special interest for biologist to observe various features of the living bodies. In the current study we observed the foliar epidermal micro‐morphological characters of 44 grass species using SEM and Light microscopy to assess their taxonomic utility for taxonomists in the identification process. The aim of this study is to use the foliar epidermal structural variations in both upper and lower surfaces for identification of grasses. Significant diversity was observed in both qualitative and quantitative characters using SEM and Light microscopy. Variations were observed in stomatal number, size, guard cells shape, silica bodies, macro‐hairs, micro‐hairs, epidermal cell number, subsidiary cells, prickles, hooks, papillae, and short and long cells. A taxonomic key is prepared using these variations for the identification of grass species. Based on these findings, SEM and Light microscopy of foliar epidermal features can be of special interest for taxonomists in the identification of complex grass taxa.