How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question.

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.

Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy.

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

Imaging the Plant Cytoskeleton by High-Pressure Freezing and Electron Tomography.

Electron tomography (ET) imaging of high-pressure frozen/freeze-substituted samples provides a unique opportunity to study structural details of organelles and cytoskeletal arrays in plant cells. In this chapter, we discuss approaches for sample preparation by cryofixation at high pressure, freeze substitution, and resin embedding. We also include pipelines for data collection for electron tomography at ambient temperature, tomogram calculation, and segmentation.

High-Pressure Freezing Followed by Freeze Substitution: An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking.

A major goal of structural biologists is to preserve samples as close to their living state as possible. High-pressure freezing (HPF) is a state-of-art technique that freezes the samples at high pressure (~2100 bar) and low temperature (-196 °C) within milliseconds. This ultrarapid fixation enables simultaneous immobilization of all cellular components and preserves the samples in a near-native state. This facilitates the study of dynamic processes in Golgi apparatus organization and membrane trafficking. The work in our laboratory shows that high-pressure freezing followed by freeze substitution (FS), the introduction of organic solvents at low temperature prior to plastic embedding, can better preserve the structure of Golgi apparatus and Golgi-associated vesicles. Here, we present a protocol for freezing monolayer cell cultures on sapphire disks followed by freeze substitution. We were able to use this protocol to successfully study Golgi organization and membrane trafficking in HeLa cells. The protocol gives decidedly better preservation of Golgi apparatus and associated vesicles than conventional chemically fixed preparation and as a plastic embedded preparation can be readily extended to 3D electron microscopy imaging through sequential block face-scanning electron microscopy. The 3D imaging of a multi-micron thick organelle such as the Golgi apparatus located near the cell nucleus is greatly facilitated relative to hydrated sample imaging techniques such as cryo-electron microscopy.

Preparation of Drosophila Tissues and Organs for Transmission Electron Microscopy.

Transmission electron microscopy (TEM) is the method of choice to image the ultrastructure of cells or tissues. TEM allows the visualization of molecular complexes up to an atomic resolution. Thus, TEM data have led to important conclusions about cellular processes and supported findings obtained by functional analyses. In this chapter, we describe the preparation of Drosophila tissues for TEM and provide reliable step-by-step protocols for applying classical chemical fixation or high-pressure freezing-freeze substitution (HPF-FS) to preserve cellular structures.

Ultrastructural examination of mouse kidney glomerular capillary loop by sandwich freezing and freeze-substitution.

Sandwich freezing is a method of rapid freezing by sandwiching specimens between two metal disks and has been used for observing exquisite the close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed animal and human tissues. In the present study, this method was applied to observe the fine structure of mouse glomerular capillary loops. Morphometry was then performed, and the results were compared with the data obtained by an in vivo cryotechnique, which may provide the closest ultrastructure to the native state of living tissue. The results show that the ultrastructure of glomerular capillary loops obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation was close to that of the ultrastructure obtained by in vivo cryotechnique not only in the quality of cell image but also in quantitative morphometry. They indicate that the ultrastructure obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation may more closely reflect the living state of cells and tissues than conventional chemical fixation and dehydration at room temperature and conventional rapid freezing-freeze-substitution of excised tissues without glutaraldehyde fixation. Sandwich freezing-freeze-substitution techniques should be used routinely as a standard method for observing the close-to-native ultrastructure of biological specimens.

Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.

Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.

High-Pressure Freezing and Transmission Electron Microscopy to Visualize the Ultrastructure of the C. auris Cell Wall.

Transmission electron microscopy (TEM) is the main technique used to study the ultrastructure of biological samples. Chemical fixation was considered the main method for preserving samples for TEM; however, it is a relatively slow method of fixation and can result in morphological alterations. Cryofixation using high-pressure freezing (HPF) overcomes the limitations of chemical fixation by preserving samples instantly. Here, we describe our HPF methods optimized for visualizing Candida auris at the ultrastructural level.

NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.

The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.

Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution.

High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

High-throughput screening of mitotic mammalian cells for electron microscopy using classic histological dyes.

We introduce a new workflow that allows screening and selection of staged mammalian cells in mitosis prior to subsequent electron microscopy. We mainly describe four improved steps of specimen preparation. Firstly, we describe a method to efficiently enrich mammalian cells and attach them to sapphire discs; secondly, we report on the use of 3D-printed containers to seed cells on coated sapphire discs for high-pressure freezing; thirdly, we take advantage of a specimen carrier that allows for an upside-down placing of sapphire discs without a second carrier or spacer ring to close the "sandwich"; and fourthly, we use histological dyes to stain DNA/chromatin during freeze-substitution. Out of 14 tested histological dyes, we routinely use four of them for visual inspection of mitotic cells by light microscopy. Applying this streamlined workflow, HeLa cells at different stages of mitosis can be selected for further ultrastructural analysis. The practical aspects of this approach will be discussed herein.

Freeze substitution Hi-C, a convenient and cost-effective method for capturing the natural 3D chromatin conformation from frozen samples.

Chromatin interactions functionally affect genome architecture and gene regulation, but to date, only fresh samples must be used in High-through chromosome conformation capture (Hi-C) to keep natural chromatin conformation intact. This requirement has impeded the advancement of 3D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time. Here, we develop a freeze substitution Hi-C (FS-Hi-C) technique that overcomes the need for fresh samples. FS-Hi-C can be used with samples stored in liquid nitrogen (LN2): the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution. After confirming that the FS step preserves the natural chromosome conformation during sample thawing, we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum. Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains, we found that FS-Hi-C outperforms the in situ Hi-C in terms of library quality, reproducibility, and valid interactions. Thus, FS-Hi-C will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.

Characterisation of early ultrastructural changes in the cerebral white matter of CADASIL small vessel disease using high-pressure freezing/freeze-substitution.

AIMS: The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease. METHODS: We used high-pressure freezing and freeze substitution (HPF/FS) in combination with high-resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3R169C ) mice aged 4-15 months to study white matter lesions in the corpus callosum. RESULTS: We first optimised the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months, then vesiculation of the inner tongue and myelin sheath thinning at 15 months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age. CONCLUSION: We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism.

Sandwich freezing device for rapid freezing of viruses, bacteria, yeast, cultured cells and animal and human tissues in electron microscopy.

We have been using sandwich freezing of living yeast and bacteria followed by freeze-substitution for observing close-to-native ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells and human tissues have been found to give excellent preservation of ultrastructure of cells and tissues. These studies, however, have been conducted using a handmade sandwich freezing device and have been limited in a few laboratories. To spread the use of this method to other laboratories, we fabricated and commercialized a new sandwich freezing device. The new device is inexpensive, portable and sterilizable. It can be used to rapid-freeze viruses, bacteria, yeast, cultured cells and animal and human tissues to a depth of 0.2 mm if tissues are prefixed with glutaraldehyde. The commercial availability of this device will expand application of rapid freezing to wide range of biological materials.

High-Pressure Freezing and Freeze Substitution for Transmission Electron Microscopy Imaging and Immunogold-Labeling.

Electron microscopy enables the unbiased imaging of organelles and cellular structures at nano-meter scale resolution. The combination of cryofixation/freeze-substitution methods with other imaging techniques such as correlative light and electron microscopy (CLEM), electron tomography (ET), and immunogold-labeling provides unique opportunities to understand structural changes associated with cellular processes. This chapter presents the main steps in the preparation of Arabidopsis thaliana roots, cotyledons, anthers, and developing seeds by high-pressure freezing and freeze-substitution for structural analysis and immunogold-labeling using transmission electron microscopy.

Electron tomography and immunogold labeling of plant cells.

Electron microscopy enables the imaging of organelles and macromolecular complexes within cells at nanometer scale resolution. Electron tomography of biological samples, either in vitrified ice or fixed and embedded in resin, provides three-dimensional structural information of relatively small volumes (a few cubic microns) of cells at axial resolutions of 1-7nm. This chapter discusses approaches for plant sample preparation by high-pressure freezing/freeze-substitution and resin-embedding for electron tomography and immunogold labeling using transmission electron microscopy.

An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM.

Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

Preparation of Cultured Cells Using High-Pressure Freezing and Freeze Substitution for Subsequent 2D or 3D Visualization in the Transmission Electron Microscope.

Transmission electron microscopy (TEM) is an invaluable technique used for imaging the ultrastructure of samples, and it is particularly useful when determining virus-host interactions at a cellular level. The environment inside a TEM is not favorable for biological material (high vacuum and high energy electrons). Also biological samples have little or no intrinsic electron contrast and rarely do they naturally exist in very thin sheets, as is required for optimum resolution in the TEM. To prepare these samples for imaging in the TEM therefore requires extensive processing which can alter the ultrastructure of the material. Here we describe a method which aims to minimize preparation artifacts by freezing the samples at high pressure to instantaneously preserve ultrastructural detail, then rapidly substituting the ice with resin to provide a firm matrix which can be cut into thin sections for imaging. Thicker sections of this material can also be imaged and reconstructed into 3D volumes using electron tomography.

Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy.

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View®) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.