Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement

Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.     

Light microscopy and scanning electron microscopy of the In vitro evagination process of Echinococcus multilocularis protoscolices

Marchiondo A. A. and Andersen F. L. 1984. Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices. International Journal for Parasitotogy14:151–157. During histogenesis of the protoscolices of Echinococcus multilocularis, the apical portion of the protoscolex consisting of the suckers, rostellum and hook region develops as an introversion and invagination within the tissue of the basal portion. In vitro incubation of protoscolices in evagination fluid stimulates the emergence of the apical portion. The initiation of evagination is first detected by a surface change in the basal portion. The smooth contour of this surface which lacks microtriches becomes transformed into tegumental indentations that form transverse and longitudinal furrows within the basal tegument as the protoscolices contract and expand, respectively. An orifice formed at the site or junction where the apical portion is invaginated begins to expand laterally in order to allow emergence of the suckers. The hooks are arranged within the invaginated protoscolex with blades directed towards the basal orifice, the handles directed towards the peduncle and the guards directed laterally. This arrangement persists throughout the evagination of the suckers and rostellum until the apical dome of the hook region emerges, thereby rotating the blades laterally in the direction of the peduncle and rotating the handles and guards medially to assume a coronal arrangement. Evagination is an asynchronous event and therefore allows observation of individual protoscolices in various stages of emergence.     

Fluorescent labeling and modification of proteins

This review provides an outline for fluorescent labeling of proteins. Fluorescent assays are very diverse providing the most sensitive and robust methods for observing biological processes. Here, different types of labels and methods of attachment are discussed in combination with their fluorescent properties. The advantages and disadvantages of these different methods are highlighted, allowing the careful selection for different applications, ranging from ensemble spectroscopy assays through to single-molecule measurements.     

In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy

Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell-localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl₂, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT-PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.     

The number and distribution pattern of Golgi bodies in cells of Micrasterias (Conjugates, Chlorophyta) examined by fluorescence microscopy and electron microscopy

The number and the distribution pattern of Golgi bodies in cells of Micrasterias americana and Micrasterias crux-melitensis were examined both by fluorescence microscopy and by electron microscopy. Golgi bodies intensely absorbed the fluorescent dye DiOC6(3) and strongly radiated fluorescent light. The number of Golgi bodies nearly doubled before septum formation, and half of the Golgi bodies entered each sister cell. Many Golgi bodies migrated from the non-growing half-cell to the growing half-cell where new cell walls were actively being synthesized. Most Golgi bodies were not in contact with chloroplasts in the growing half-cell. Half of the Golgi bodies moved back to the non-growing half-cell 6–12 h after completion of the new half-cell. Golgi bodies were in contact with the surfaces of chloroplasts 12 h after full growth.     

Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy

Virus assembly occurs in a complex environment and is dependent upon viral and cellular components being properly correlated in time and space. The simplicity of the flock house virus (FHV) capsid and the extensive structural, biochemical and genetic characterization of the virus make it an excellent system for studying in vivo virus assembly. The tetracysteine motif (CCPGCC), that induces fluorescence in bound biarsenical compounds (FlAsH and ReAsH), was genetically inserted in the coat protein, to visualize this gene product during virus infection. The small size of this modification when compared to those made by traditional fluorescent proteins minimizes disruption of the coat proteins numerous functions. ReAsH not only fluoresces when bound to the tetracysteine motif but also allows correlated electron microscopy (EM) of the same cell following photoconversion and osmium staining. These studies demonstrated that the coat protein was concentrated in discrete patches in the cell. High pressure freezing (HPF) followed by freeze substitution (FS) of infected cells showed that these patches were formed by virus particles in crystalline arrays. EM tomography (EMT) of the HPF/FS prepared samples showed that these arrays were proximal to highly modified mitochondria previously established to be the site of RNA replication. Two features of the mitochondrial modification are 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle.     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.     

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

To genuinely understand how complex biological structures function, we must integrate knowledge of their dynamic behavior and of their molecular machinery. The combined use of light or laser microscopy and electron microscopy has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, is often referred to as ‘correlative microscopy’. Correlative imaging allows researchers to gain additional novel structure–function information, and such information provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or ‘combined’) microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches. In this topical review, we provide a brief historical perspective of correlative microscopy and an in-depth overview of correlative sample-preparation and imaging methods presently available, including future perspectives on the trend towards integrative microscopy and microanalysis.     

Light and electron microscopy of the hepatopancreas of the isopod Acellus intermedius

Summary Hepatopancreas from Acellus intermedius, a small freshwater isopod, was examined by light and electron microscopy. Of particular interest are the two cell types present in the hepatopancreas. Their ultrastructural features indicate that the major role of the large alpha cells is to absorb material from the lumen as well as secrete materials into the lumen while the major role of the small beta cells is to store the absorbed materials as well as break down stored materials before transport.     

The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts. International journal for Parasitology4: 139–142. Cysts of 4 strains of non-pathogenic Naegleria gruberi and 5 strains of pathogenic Naegleria fowleri were examined in the scanning electron microscope. Excystment of the Naegleria gruberi amoebae occurred via preformed exit pores in the cyst wall. Similar structures were not found in the cysts of Naegleria fowleri, and excystment occurred by rupture of the cyst wall. The sequence of cyst wall rupture is illustrated for one of the pathogenic strains.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

Crystallisation of CP43, a chlorophyll binding protein of photosystem II: an electron microscopy analysis of molecular packing

Microcrystals of the chlorophyll binding protein, CP43, isolated from spinach thylakoid membranes have been studied by electron microscopy both in negative stain and in vitreous ice. Image analyses of three characteristic views show that the crystals are built of five different layers perpendicular to the c-axis. Each layer consists of different orientations of the CP43 protein. The unit cell derived from the end-on view (looking down the c-axis) shows an angle of 120 degrees, suggesting a threefold rotational symmetry. Both negative staining and cryo data are consistent with a hexagonal crystal lattice. Interpretation of the arrangement of the CP43 protein within this crystal lattice can be made based on 8- and 9-A electron crystallographic structures previously published that provide a model for the organisation of the transmembrane helices of CP43. Overall the analysis presented is consistent with X-ray diffraction data obtained from larger CP43 crystals and forms a framework on which to base further structural studies of this chlorophyll binding protein.