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Freeze substitution is a crucial technique in electron microscopy, enabling the preservation of biological specimen ultrastructure. However, existing tools are primarily designed for 3 mm carriers, limiting their compatibility with larger carriers such as CryoCapsules or 6 mm carriers. In this study, we developed a novel freeze substitution tool, simply named the FS-basket, to address the challenges associated with embedding samples vitrified in larger carriers. The tool comprises a mesh with pore sizes smaller than the sample chunks, deposited below the carrier to maintain a tight seal while allowing for easy fluid exchange. [ABSTRACT FROM AUTHOR]

This study explores the efficacy and reliability of high-pressure freezing (HPF) as a sample preparation technique for electron microscopy (EM) analysis across a diverse range of biological samples. Utilizing the HPM Live µ technology, based on the historical hydraulic HPM010 from BalTec, we demonstrate the reliability of our industrial equipment to achieve the critical parameters necessary for vitrification (2076 bars, cooling rate above 2000 K/s). By directly measuring physical values within the HPF chamber, we evaluate the proper functioning of the equipment, contributing to the technique's reliability. A meticulous approach was adopted for each sample type, acknowledging the uniqueness of each specimen, and associating the final sample analysis with its HPF curve, aiding in protocol optimization. Samples including human cell pellets, cell monolayer, mouse brain and liver biopsies, and Arabidopsis thaliana root and seedlings were processed for EM analysis following HPF. The ultrastructure of each sample type was rigorously examined, revealing homogeneous preservation and minimal ice nucleation artefacts. Overall, our work underscores the robustness and versatility of our HPM Live µ in preserving biological ultrastructure, offering valuable insights for researchers employing EM techniques in diverse biological studies. [ABSTRACT FROM AUTHOR]

The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium‐containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well‐established light and advanced electron microscopy techniques in this system, a correlative approach integrating both methods with near‐native ultrastructural preservation is currently lacking. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D‐correlative light and electron microscopy. This approach includes high‐pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens. [ABSTRACT FROM AUTHOR]

In this study, we aimed to document the freshwater symbiotic interactions along with the impact of the abiotic environment and anthropogenic effects on the functionality of freshwater organisms. Symbiotic green hydra (Z) and free-living brown hydra (S), either separately or both species together, were treated with the herbicide norflurazon in concentrations of 2 × 10−6 mol/L (N6) and 2 × 10−7 mol/L (N7) for 72 h. Also, hydras were treated with both norflurazon and UV radiation at a wavelength of 254 nm for 2 min or were irradiated only. The next part of the experiment was performed in the same way but with added suspensions of isolated endosymbiotic alga, free-living alga, or both algae together. Mortality, migration, tentacle and tissue damage, changes in the thickness of the mesoglea of hydras, and clustering of algae were monitored. Green hydra generally showed lower rates of migration, and mortality was observed only in green hydra exposed to UV radiation. Tentacle damage was more pronounced in green hydra and included a specific fork-like structure. The use of cryofixation and TEM enabled us to partly elucidate the effect of clustering of algae. In summary, our study provides new insights into the influence of different environmental stressors and their combination on symbiotic and free-living freshwater hydras and algae and a better understanding of interactions in freshwater ecosystems. [ABSTRACT FROM AUTHOR]

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures. [ABSTRACT FROM AUTHOR]

The microcosm is a laboratory method frequently used in ecological studies related to population and food web interactions and environmental dynamics. It simultaneously brings into interaction different species in the same controlled laboratory experimental area and provides an opportunity for modeling and reconstruction of relationships in the natural biocenoses and ecosystems. We applied that approach to determine and improve our understanding of predator–prey interactions in different freshwater environments. The inhabitants of the microcosms were isolated endosymbiotic microalga Desmodesmus subspicatus (Chlorophyceae) (Chodat) Hegewald et Schmidt (CZ), green hydras, freshwater turbellarians, and large water fleas. Experiments were performed in five replicates, at 25 °C and 13.5 °C, with fed and hungry predators, respectively. Herein, we proposed a mechanism for microalgal hunting net formation in the freshwater microcosm. Ultrastructural visualization of the endosymbiotic microalgae revealed rod-like structures on the cell wall surface, structures that could possibly fit together and interconnect, suggesting the possibility of microalgal hunting net formation. Interspecific cooperation between isolated microalgae and turbellarians resulted in stronger hunting net formation in preying upon water fleas. This study contributes to the diversity of species interactions and shows the producers as a top link, as opposed to what is generally considered as a basic link in the food web, and presents the microalgae as triggers of the dynamics in the freshwater microcosm. [ABSTRACT FROM AUTHOR]

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell. [ABSTRACT FROM AUTHOR]

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.

The microcosm is a laboratory method frequently used in ecological studies related to population and food web interactions and environmental dynamics. It simultaneously brings into interaction different species in the same controlled laboratory experimental area and provides an opportunity for modeling and reconstruction of relationships in the natural biocenoses and ecosystems. We applied that approach to determine and improve our understanding of predator–prey interactions in different freshwater environments. The inhabitants of the microcosms were isolated endosymbiotic microalga Desmodesmus subspicatus (Chlorophyceae) (Chodat) Hegewald et Schmidt (CZ), green hydras, freshwater turbellarians, and large water fleas. Experiments were performed in five replicates, at 25 °C and 13.5 °C, with fed and hungry predators, respectively. Herein, we proposed a mechanism for microalgal hunting net formation in the freshwater microcosm. Ultrastructural visualization of the endosymbiotic microalgae revealed rod-like structures on the cell wall surface, structures that could possibly fit together and interconnect, suggesting the possibility of microalgal hunting net formation. Interspecific cooperation between isolated microalgae and turbellarians resulted in stronger hunting net formation in preying upon water fleas. This study contributes to the diversity of species interactions and shows the producers as a top link, as opposed to what is generally considered as a basic link in the food web, and presents the microalgae as triggers of the dynamics in the freshwater microcosm.

PurposeSeveral properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques.MethodsA simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed.ResultsAn inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.ConclusionsA simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

Abstract Electron microscopy (EM) provides the necessary resolution to visualize the finer structures of nervous tissue morphology, which is important to understand healthy and pathological conditions in the brain. However, for the interpretation of the micrographs the tissue preservation is crucial. The quality of the tissue structure is mostly influenced by the post mortem interval (PMI), the time of death until the preservation of the tissue. Therefore, the aim of this study was to optimize the preparation-procedure for the human frontal lobe to preserve the ultrastructure as well as possible despite the long PMIs. Combining chemical pre- and post-fixation with cryo-fixation and cryo-substitution (“hybrid freezing”), it was possible to improve the preservation of the neuronal profiles of human brain samples compared to the “standard” epoxy resin embedding method. In conclusion short PMIs are generally desirable but up to a PMI of 16 h the ultrastructure can be preserved on an acceptable level with a high contrast using the “hybrid freezing” protocol described here.

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm2) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition. [ABSTRACT FROM AUTHOR]

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant. It appears that damage is associated with the formation of ice per se, even at cooling rates that are optimal for the cryopreservation of isolated chondrocytes. We then showed that current methods of cartilage cryopreservation involve the nucleation and growth of ice crystals within the chondrons rather than ice being restricted to the surrounding acellular matrix. This finding established the need to avoid the crystallization of ice—in other words, vitrification. Song and his colleagues have published a vitrification method that is based on the use of one of Fahy's vitrification formulations. We confirmed the effectiveness of this method but found it to be very dependent on ultra rapid warming. However, we were able to develop a 'liquidus-tracking' method that completely avoids the crystallization of ice and does not require rapid warming. The ability of cartilage preserved in this way to incorporate sulphate into newly synthesized glycosaminoglycans (GAGs) approached 70% of that of fresh control cartilage. In this method the rates of cooling and warming can be very low, which is essential for any method that is to be used in Tissue Banks to process the bulky grafts that are required by orthopaedic surgeons. Work is continuing to refine this method for Tissue Bank use. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

High-pressure freezing/freeze substitution has been used to preserve biological samples for ultrastructure study instead of chemical fixation. For most plant samples, the water content is too high and cannot be properly preserved during cryofixation. Additionally, the cell wall is a barrier that prevents the substitution of water with the resin. In this chapter, we will discuss modified high-pressure freezing and subsequent processing protocols based on our routinely used methodology for examining Arabidopsis seeds in transmission electron microscopy and electron tomography., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plastination is a late 20th century preservation methodology which replaces tissue fluid within a specimen with a curable polymer, such as silicone. Plastination yields superb, beautiful, well‐preserved specimens each with their own unique qualities. Silicone polymer is used around the world to preserve macroscopic cadavers or portions/organs thereof. Plastination was conceived by Dr. Gunther von Hagens, Universität Heidelberg, Heidelberg, Germany prior to 1977. Silicone polymer was the primary polymer which emerged initially for plastination. The Biodur® line of silicone polymer and additives was chosen and manufactured because it has consistently produced the best plastinates since the inception of plastination. Since the discovery of silicone, generic and similar silicone polymers are known and used around the World by many industries and used in numerous products. The plastination process has four steps: Specimen preparation, Specimen dehydration and degreasing, Vacuum‐forced impregnation of specimens and Specimen hardening. [ABSTRACT FROM AUTHOR]

For 20 years, the cold temperature/S10/von Hagens' plastination technique was used to preserve biological specimens without challenge. It became the "gold standard" for preservation of beautiful, dry biological specimens. Near the end of the 21st century, a group from the University of Michigan and environs and Dow Corning™, USA, combined silicone ingredients, similar to the von Hagens' plastination products, however in a different sequence. The new polymer (Cor‐tech) was combined with the cross‐linker to design the "impregnation mix" which would invade the cellular structure of the specimen and yet was stable at room temperature. Later, curing would be by application of the catalyst onto the impregnated specimen. This unique sequencing of products would become the "Room temperature/Dow Corning™/Corcoran—Silicone plastination technique." The results of this room temperature technique provided similar plastinates, beautiful and practical for demonstration, containing no toxic chemical residues and forever preserved. As the name implies, impregnation of this silicone mix could be done at room temperature, without having to be kept cold. Both processes (cold and room temperature) required the same four basic steps for plastination. As well, both processes used similar basic polymers and additives to produce plastinates. However, they were combined in a different sequence. Cold temperature combines polymer and catalyst/chain extender, which is not stable and therefore must be kept colder than −15°C, while room temperature combines polymer with cross‐linker which is stable, and likely forever. [ABSTRACT FROM AUTHOR]

In 1977, plastination was unveiled, which replaced tissue fluid with a curable polymer. Today preservation via plastination of various animal and plant tissues, organs, and whole bodies is an extremely useful technique to display such and help educate vast arrays of both allied science students and the lay public across the planet. The diversity of applications of plastination techniques seems to be without limits. In fact, the only real limitation to plastination is one's imagination! The size of plastinates during the early years of plastination was comparatively small and dictated primarily by the size of the available plastination kettle/chamber, 35 L Heidelberg plastination kettle (49 cm H × 34.5 cm diam.). In the 1990s larger chambers were designed and slowly became available:150–210 cm (long) × 65–80 cm (wide) and 83–92 cm (high). Today a few large vacuum chambers are in service which will accommodate whole bodies of man and domestic or exotic animals. Today, at least two gigantic chambers are available to impregnate massive specimens. These are 3.5 m × 2 m × 1.5 m (Dalian) and 4 m × 3 m × 2.2 m (Guben). Also, the need for larger quantities of acetone and impregnation mix, not to mention the great increase in specimen preparation time, makes this a major investment. The "cold temperature process" is used to impregnate these massive creations. The room temperature technique could be used. The same four plastination steps are necessary for larger and massive specimens. Besides their tremendous size, the slippery silicone polymer is a reckoning force. [ABSTRACT FROM AUTHOR]

Summary: High‐pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high‐pressure freezer. We explored the potential of a 10 μm thin polyester filter membrane (PE‐filter) as a versatile sample substrate for high‐pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE‐filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE‐filter. These microorganism‐loaded PE‐filters were used for high‐pressure freezing, freeze‐substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross‐sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa‐cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism‐loaded PE‐filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze‐substituted PE‐filters samples with scanning electron microscopy, demonstrates the versatility of the PE‐filter as a sample substrate for a wide array of microorganisms. Lay Description: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology‐changes due to drying artefacts or the impact of chemical stabilisers, high‐pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to –196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology‐destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 μm in optimal cases). High‐pressure freezing is regarded as the gold‐standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high‐pressure freezer and their transfer into the high‐pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 μm thin polyester filter membrane (PE‐filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE‐filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface‐adhering higher cells were able to grow directly on the PE‐filter. These microorganism‐loaded PE‐filters could be directly used for high‐pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross‐sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa‐cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism‐loaded PE‐filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze‐substituted PE‐filters samples with scanning‐electron microscopy, demonstrates the versatility of the PE‐filter as a sample substrate for a wide array of microorganisms. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Scanning transmission electron microscopic (STEM) tomography of high-pressure frozen, freeze-substituted semi-thin sections is one of multiple approaches for three-dimensional recording and visualization of electron microscopic samples. Compared to regular TEM tomography thicker sample sections can be investigated since chromatic aberration due to inelastic scattering is not a limit. The method is ideal to investigate subcellular compartments or organelles such as synapses, mitochondria, or microtubule arrangements. STEM tomography fills the gap between single-particle electron cryo-tomography, and methods that allow investigations of large volumes, such as serial block-face SEM and FIB-SEM. In this article, we discuss technical challenges of the approach and show some applications in cell biology. It is ideal to use a 300-kV electron microscope with a very small convergence angle of the primary beam (“parallel” beam). These instruments are expensive and tomography is rather time consuming, and therefore, access to such a high-end microscope might be difficult. In this article, we demonstrate examples of successful STEM tomography in biology using a more standard 200-kV microscope equipped with a field emission tip. [ABSTRACT FROM AUTHOR]

Self-Pressurised Rapid Freezing (SPRF), an easy-to-use and low-cost alternative cryo-fixationmethod, was evaluated based on a comparative analysis of the ultrastructure of spermatozoa of the nematodes Acrobeles complexus and Caenorhabditis elegans. Sealed copper tubes, packed with active nematodes in water, were plunged into nitrogen slush, a semi-solid form of nitrogen. The water inside the capillary copper tube expands upon cooling due to the formation of hexagonal ice, thereby generating high pressure intrinsically for cryo-fixation of the sample. For sperm cells cryo-fixed by SPRF, the preservation of the ultrastructure was comparable to that achieved with high pressure freezing. This was evidenced by the clear details in mitochondria, membranous organelles and cytoskeleton in the pseudopod. It was demonstrated that SPRF fixation did not destroy antigenicity, based on the results of the immunolocalisation of the major sperm protein in both species. In conclusion, SPRF is a low-cost alternative cryo-fixation method for nematodes. [ABSTRACT FROM AUTHOR]