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A new approach for cryofixation by high-pressure freezing
Abstract
A newly designed high-pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.
... Frequently, it is easier and sufficient for CLEM to image the sample at room temperature, therefore HPF samples can be chemically fixed at very low temperatures using a technique called freeze substitution so the fine cellular details can be preserved. The benefits and challenges of HPF and freeze substitution have been extensively described in the literature (Humbel, 2009;Humbel & Schwarz, 1989;McDonald, 2014;Studer, Graber, Al-Amoudi, & Eggli, 2001;Studer, Humbel, & Chiquet, 2008). In this example, we will describe how to prepare cultured cells for CLEM using HPF in combination with (automatic) freeze substitution (AFS) sample processing. ...
... The gold standard for fixation of cells and tissues for immuno-labeling is high-pressure freezing followed by freeze substitution and resin embedding (Humbel, 2009;Humbel & Schwarz, 1989;Studer et al., 2001Studer et al., , 2008. Alternatively, frozen samples can be subjected to freeze substitution, rehydration and Tokuyasu-cryo-sectioning (Ripper, Schwarz, & Stierhof, 2008;van Donselaar, Posthuma, Zeuschner, Humbel, & Slot, 2007). ...
... High-pressure freezing is a specialized sample preservation technique that can yield high fidelity sample preservation when executed properly (Humbel, 2009;Humbel & Schwarz, 1989;McDonald, 2014;Studer et al., 2001Studer et al., , 2008. Unfortunately, high-pressure freezing is a technique that often needs optimization over several experiments which can make it a frustrating technique to establish. ...
... This process implants cesium atoms into the sample, which leads to higher secondary ion yields and thus better signal-to-noise ratio. Fig. 1c shows isotopic maps of 12 C, 16 O, 12 C 14 N, 31 P, 32 S and 34 S of the PMN indicated with a white rectangle in Fig. 1a, and measured using the cesium(Cs)-source. Note that when using the nano-SIMS Cs-source, elements such as C, O and N can be imaged, privileging this source to investigate lighter elements in single cells. ...
... Note that when using the nano-SIMS Cs-source, elements such as C, O and N can be imaged, privileging this source to investigate lighter elements in single cells. The basic morphology of the PMN is clearly recognizable in the maps of 12 C, 16 O and 12 C 14 N. Although the size of a single pixel in the isotope maps is 20 nm, the actual resolution was estimated at approx. ...
... Region indicated with a white rectangle indicates the PMN scanned with nano-SIMS. b) TEM image of a single PMN from the same control culture c) distribution of 12 C, 16 O, 12 C 14 N, 28 Si, 31 P, Esi (electron secondary ionization), 32 S, 34 S in a single PMN from control culture (cesium source, 10 Â 10 mm 2 , 512 Â 512 pixels, 20 nm/pixel, 100 planes, 2 pA, pre-sputtered for 15 min with 10 pA), the cell membrane of the PMN is indicated with a white dashed line, d) distribution of 28 Si, 39 planes, no pre-sputtering). In c) and d), natural abundance of the measured isotope is given in the lower left corner of each map. ...
Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryo-substituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 μm for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.
... 4.5.6 TEM microscopy for P. tricornutum morphotype characterization 4.5.6.1 High pressure freezing (HPF) and freeze substitution (FS) HPF was performed with the freezer HPF-EM PACT I Leica-microsystems (Studer et al., 2001). Prior to freezing, P. tricornutum suspension-cultured cells were treated 2 hours with 100 mM mannitol as cryoprotectant diluted in culture medium at room temperature. ...
... After high-pressure freezing, samples were transferred to a freeze substitution automate (AFS, Leica, http://www.leica-microsystems.com) pre-cooled to -140°C. Freeze substitution conditions followed a modified procedure from Studer et al. (2001). Samples were substituted in anhydrous acetone with 0.5% uranyl acetate at -90°C for 96 h. ...
La N-glycosylation est un événement co- et post-traductionnel majeur de la synthèse protéique chez les eucaryotes. Actuellement, peu d’informations concernant ce processus sont disponibles chez les microalgues. Dans ces travaux, nous avons poursuivi l’exploration de la voie de N-glycosylation de la diatomée modèle P. tricornutum en focalisant spécifiquement notre travail sur la caractérisation de trois α1,3-fucosyltransférases putatives. Nos analyses ont permis de remettre en cause l’annotation pour deux des gènes codant pour ces fucosyltransférases et d’en proposer de nouvelles. Afin de réaliser l’étude de la localisation subcellulaire de ces glycosyltransférases, nous avons utilisé une stratégie de sur-expression de ces protéines fusionnées avec une étiquette. Les observations menées par des approches de microscopie confocale et de microscopie électronique à transmission ont permises de mettre en évidence la localisation subcellulaire golgienne de ces enzymes, ainsi que de trois autres glycosyltransférases utilisées comme témoin dans ce travail. Cette étude a permis de réaliser la première localisation subcellulaire de glycosyltransférases chez les microalgues. P. tricornutum est une diatomée pléïomorphique qui possède trois morphotypes majeurs différents appelés ovale, fusiforme et triradié. Le passage d’un morphotype à un autre dépend fortement des conditions environnementales. Cependant, peu d’informations sont disponibles concernant les mécanismes et la signification physiologique de cette morphogenèse. Afin d’apporter de nouvelles réponses, nous avons réalisé une analyse comparative du transcriptome des trois morphotypes par une approche transcriptomique à haut débit appelée RNASeq pour RNA-Sequencing. Enfin, en plus d’élargir les connaissances sur la morphogénèse de P. tricornutum, les données générées ont également permis d’identifier de nouveaux gènes de référence utilisables pour des analyses de qRT-PCR.
... The bacteria were pelleted by centrifugation (5,000 rpm for 5 min) and the supernatant was discarded. Bacteria were then cryopreserved by high-pressure freezing (HPF) as described in Studer et al. (2001) using 1.4 x 0.1 mm membrane carriers (Leica Microsystems, Vienna) coated with L-α-phosphatidylcholine (Fluka, Buchs, Switzerland) (Studer et al., 2001). Acetone containing 2 % osmium tetroxide, 0.1% uranyl acetate, 0.2% ruthenium hexamine trichloride (RHT) and a total of 4% H 2 O served as medium for freeze substitution (FS). ...
... The bacteria were pelleted by centrifugation (5,000 rpm for 5 min) and the supernatant was discarded. Bacteria were then cryopreserved by high-pressure freezing (HPF) as described in Studer et al. (2001) using 1.4 x 0.1 mm membrane carriers (Leica Microsystems, Vienna) coated with L-α-phosphatidylcholine (Fluka, Buchs, Switzerland) (Studer et al., 2001). Acetone containing 2 % osmium tetroxide, 0.1% uranyl acetate, 0.2% ruthenium hexamine trichloride (RHT) and a total of 4% H 2 O served as medium for freeze substitution (FS). ...
Non-encapsulated Streptococcus pneumoniae often possess two genes, aliB-like ORF 1 and aliB-like ORF 2, in place of capsule genes. AliB-like ORF 1 is thought to encode a substrate binding protein of an ABC transporter which binds peptide SETTFGRDFN, found in 50S ribosomal subunit protein L4 of Enterobacteriaceae. Here, we investigated the effect of binding of AliB-like ORF 1 peptide on the transcriptome and proteome of non-encapsulated pneumococci. We found upregulation of gene expression of a metacaspase and a gene encoding N-acetylmuramoyl-L-alanine amidase, both of which are proposed to be involved in programmed cell death in prokaryotic cells. Proteome profiling indicated upregulation of transcriptional regulators and downregulation of metabolism-associated genes. Exposure to the peptide specifically triggered death in pneumococci which express AliB-like ORF 1, with the bacteria having an apoptotic appearance by electron microscopy. We propose that binding of the AliB-like ORF 1 peptide ligand by the pneumococcus signals a challenging environment with hostile bacterial species leading to death of a proportion of the pneumococcal population.
... High pressure freezing (HPF) and freeze substitution (FS). HPF was performed with the freezer HPF-EM PACT I Leica-microsystems 76 . Prior to freezing, P. tricornutum suspension-cultured cells were treated 2 hours with 100 mM mannitol as cryoprotectant diluted in culture medium at room temperature. ...
... After high-pressure freezing, samples were transferred to a freeze substitution automate (AFS, Leica, http://www.leica-microsystems.com) pre-cooled to −140 °C. Freeze substitution conditions followed a modified procedure from ref. 76 . Samples were substituted in anhydrous acetone with 0.5% uranyl acetate at −90 °C for 96 h. ...
Phaeodactylum tricornutum is the most studied diatom encountered principally in coastal unstable environments. It has been hypothesized that the great adaptability of P. tricornutum is probably due to its pleomorphism. Indeed, P. tricornutum is an atypical diatom since it can display three morphotypes: fusiform, triradiate and oval. Currently, little information is available regarding the physiological significance of this morphogenesis. In this study, we adapted P. tricornutum Pt3 strain to obtain algal culture particularly enriched in one dominant morphotype: fusiform, triradiate or oval. These cultures were used to run high-throughput RNA-Sequencing. The whole mRNA transcriptome of each morphotype was determined. Pairwise comparisons highlighted biological processes and molecular functions which are up- and down-regulated. Finally, intersection analysis allowed us to identify the specific features from the oval morphotype which is of particular interest as it is often described to be more resistant to stresses. This study represent the first transcriptome wide characterization of the three morphotypes from P. tricornutum performed on cultures specifically enriched issued from the same Pt3 strain. This work represents an important step for the understanding of the morphogenesis in P. tricornutum and highlights the particular features of the oval morphotype.
... Over the past decade, real-time imaging and 3D electron tomography (ET) have increasingly replaced conventional light microscopy and immunofluorescence microscopy (LM and IF) and 2D transmission electron microscopy, respectively. In addition, fast freezing technologies (vitrification, high-pressure freezing, HPF) have become available to arrest cellular membrane dynamics within milliseconds, thereby providing a physiological "snapshot" of the cell without the artifacts produced by chemical fixation [19][20][21]. Fast immobilization methods, combined with (cryo) TEM tomography and 3D reconstruction allow the snap-frozen structures to be reconstructed into 3D models [22][23][24]. This led to the development of new approaches that combine light microscopy imaging with high-resolution (cryo) electron microscopy (correlative light and electron microscopy, CLEM) [25,26]. ...
... At this level of pressure, the freezing point of water is lowered down to about −20 °C, and the nucleation of ice crystals as well as their growth is slowed down. For more details about the theory behind this method, see the articles by Riehle and Höchli [19], Studer et al. [20], and Vanhecke et al. [21]. HPF is capable of freezing cells and tissue samples up to 200 μm (see Note 10). ...
Blood platelets play a central role in the arrest of bleeding and the development of thrombosis. Unraveling the complex processes of platelet biogenesis from megakaryocytes, platelet adhesion, aggregation, and secretory responses are important topics in the field of hemostasis and thrombosis. Analysis of the ultrastructural changes that occur during these processes is essential for understanding the rapid membrane dynamics and has contributed substantially to our present knowledge of platelet formation and functioning. Recent developments in real-time imaging, correlative light and electron microscopy imaging (CLEM), and 3D (cryo) electron microscopy and tomography offer exciting opportunities to improve studies of the platelet adhesive responses and secretion at the ultrastructural level in a close to native environment. In this chapter we discuss and illustrate cryo preparation techniques (high-pressure freezing, vitrification), correlative LM and EM workflows, and 3D cryo-electron tomography that we apply in our current research projects.
... They were then cooled under high pressure (2000 bar) to liquid nitrogen temperature (−196 °C) within milliseconds using an EM-PACT2 machine (Leica Microsystems, Wetzlar, Germany). These conditions prevent the formation of ice crystals, which can damage cellular structures [50]. The frozen samples were then cut into thin slices (50 nm) in a cryo-ultramicrotome UC6 FC6 (Leica Microsystems, Wetzlar, Germany) and placed on a R3.5-1 holey carbon EM grid (Quantifoil, Großlöbichau, Germany). ...
The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores ; Actinobacteria , forming exospores ; Cyanobacteria , forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.
... Before fixation, ensure that the fluorescence signals are easily visible through the oculars for individual samples with non-destructive observations (stereomicroscopy or macroscopy). 1. HPF Note: HPF was done using the Leica EM-PACT1 machine (Studer et al., 2001). A detailed protocol is described in Nicolas et al. (2018). ...
Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana , which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues. Graphical abstract.
... In recent years, significant efforts have been made to overcome the size limitations of the samples that can be imaged with cryo-ET. In order to enable the proper vitrification of thicker samples, high-pressure can be applied during the freezing process, which prevents sample expansion due to crystallization ( Fig. 1) (Moor 1987;D. Studer et al. 2001). This allows complete vitrification of samples up to 200 µm (Daniel Studer et al. 2008). For high-pressure freezing, the biological sample is loaded into copper or aluminum planchette carriers with the addition of a cryoprotectant. Subsequently, the sample is frozen with 2000 atm pressure in milliseconds. If the sample is below the size ...
Animals, plants, and fungi live in a microbe-dominated world. Investigating the interactions and processes at the host-microbe interface offers insight to how bacteria influence the development, health, and disease of the host. Optimization of existing imaging technologies and development of novel instrumentation will provide the tools needed to fully understand the dynamic relationship that occurs at the host-microbe interface throughout the lifetime of the host. In this review, we describe the current methods used in cryo-electron microscopy (cryo-EM) including cryo-fixation, sample processing, FIB-SEM, and cryotomography. Further, we highlight the new advances associated with these methods that open the cryo-EM discipline to large, complex multicellular samples, like symbiotic tissues. We describe the advantages and challenges associated with correlative imaging techniques and sample thinning methods like lift-out. By highlighting recent pioneering studies in the large-volume or symbiotic sample workflows, we provide insight into how symbiotic model systems will benefit from cryo-EM methods to provide artefact-free, near-native, macromolecular-scale resolution imaging at the host-microbe interface throughout the development and maintenance of symbiosis. Cryo-EM methods have brought a deep fundamental understanding of prokaryotic biology since its conception. We propose the application of existing and novel cryo-EM techniques to symbiotic systems is the logical next step that will bring an even greater understanding how microbes interact with their host tissues.
... Successful vitrification requires rapid freezing that does not result in the formation of crystalline ice but produces amorphous ice, which is largely transparent to laser and electron beams. Two common means of vitrification include plunge freezing (Dobro et al. 2010) and high-pressure freezing (Studer et al. 2001). For plunge freezing, an EM grid, which has eukaryotic cells grown on it or a drop of suspension that contains cell fragments or prokaryotic cells deposited on it, is blotted with filter paper and then plunged into liquid ethane to vitrify the contents. ...
Electron microscopy (EM) reveals cellular ultrastructure at high definition but faces the challenges of identification of specific subcellular structures and localization of specific macromolecules, whereas fluorescence microscopy (FM) can label and localize specific molecules in cells. Correlative light and electron microscopy (CLEM) combines the advantages of both microscopic techniques. Imaging vitreous hydrated samples at cryogenic temperatures using CLEM enables observations of cellular components of interest and their cellular context in a near-native state. This cryo-CLEM approach is further strengthened by incorporation of superresolution fluorescence microscopy, which can precisely pinpoint targets on electron micrographs. Cryogenic superresolution correlative light and electron microscopy (csCLEM) is an emerging and promising imaging technique that is expected to unveil its full power in ultrastructural studies. The present review describes the logic and principles behind this technique, how the method is implemented, the prospects, and the challenges.
... A decrease in the freezing rate causes the devitrification of water and ice crystals are formed (Thompson et al., 2016). The same protocol, as plunge freezing, is used with the difference of applying a high pressure around ≈ 2000 bar and using jet-stream liquid nitrogen to ensure a fresh sample (Studer et al., 1989(Studer et al., , 2001. This protocol avoids the formation of ice crystals and is well appreciated for immunolocalization, and peptides and lipids studies (Börner et al., 2006). ...
The potential of mass spectrometry imaging (MSI) has been demonstrated in cell and tissue research since 1970. MSI can reveal the spatial distribution of a wide range of atomic and molecular ions detected from biological sample surfaces, it is a powerful and valuable technique used to monitor and detect diverse chemical and biological compounds, such as drugs, lipids, proteins, and DNA. MSI techniques, notably matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and time of flight secondary ion mass spectrometry (TOF-SIMS), witnessed a dramatic upsurge in studying and investigating biological samples especially, cells and tissue sections. This advancement is attributed to the submicron lateral resolution, the high sensitivity, the good precision, and the accurate chemical specificity, which make these techniques suitable for decoding and understanding complex mechanisms of certain diseases, as well as monitoring the spatial distribution of specific elements, and compounds. While the application of both techniques for the analysis of cells and tissues is thoroughly discussed, a briefing of MALDI-TOF and TOF-SIMS basis and the adequate sampling before analysis are briefly covered. The importance of MALDI-TOF and TOF-SIMS as diagnostic tools and robust analytical techniques in the medicinal, pharmaceutical, and toxicology fields is highlighted through representative published studies.
... Under these circumstances, the ice nucleation and distortion of specimens during solidification by freezing are minimized: intrinsically contained water turns into vitreous ice even in the absence of cryo-protectants. This results in preserving the cellular architecture (Studer et al., 2001) without requiring the use of any chemical fixatives, hence capturing a near-native, physiologically relevant state. Since this type of freezing occurs on the order of milliseconds, highly dynamic cellular events can be captured with high molecular fidelity. ...
The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.
... The cooling rates attained by plunging such specimens even into the best cryogen are insufficient to vitrify the liquid because of their geometry. That can be overcome by high-pressure freezing (HPF) [65,66], namely, by cooling the specimen while it is subjected to high pressure, about 200 MPa, of liquid nitrogen, which slows nucleation and growth rate of ice crystals. A comparison of cryo-SEM specimen preparation by plunging and by HPF in the study of EVs released from monocytes was performed by Koifman et al. [8]. ...
Many pharmaceutics are aqueous dispersions of small or large molecules, often self-assembled in complexes from a few to hundreds of molecules. In many cases, the dispersing liquid is non-aqueous. Many pharmaceutical preparations are very viscous. The efficacy of those dispersions is in many cases a function of the nanostructure of those complexes or aggregates. To study the nanostructure of those systems, one needs electron microscopy, the only way to obtain nanostructural information by recording direct images whose interpretation is not model-dependent. However, these methodologies are complicated by the need to make liquid systems compatible with high vacuum in electron microscopes. There are also issues related to the interaction of the electron beam with the specimen such as micrograph contrast, electron beam radiation damage, and artifacts associated with specimen preparation. In this article, which is focused on the state of the art of imaging self-assembled complexes, we briefly describe cryogenic temperature transmission electron microscopy (cryo-TEM) and cryogenic temperature scanning electron microcopy (cryo-SEM). We present the principles of these methodologies, give examples of their applications as analytical tools for pharmaceutics, and list their limitations and ways to avoid pitfalls in their application.
... Cryo-fixation circumvents this problem and preserves the cytoplasm in a fraction of a second; however, flawless vitrification is difficult to achieve except for the outermost micrometers (e.g., 10 μm) of tissues; in deeper layers of cells and tissues, ice crystal damage occurs. The technique of highpressure freeze fixation followed by freeze substitution greatly increases the depth and extent of high-quality fixation as described by Knoll et al. (1987), Moor (1987), Studer et al. (2001), andMcDonald et al. (2007) and since then yielded some excellent results in plant cells (e.g., Donohoe et al. 2007;Wilson and Bacic 2012;Karahara and Kang 2014;Gergely et al. 2018) and animal cells (e.g., Hess et al. 2018). ...
Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.
... In both cases, this almost necessarily implies morphological and structural changes in the sample. The use of CryoSEM has the great advantage of introducing minimal chemical changes, ensuring the samples will always be as close to their original (native) state [48][49][50][51]. Growth inside oleuropein vesicles, such as those observed through CryoSEM and shown in Figure 9, can explain both the small diameter of the nanoparticles obtained in the presence of the higher oleuropein concentrations, and their abnormal stability to both aggregation and dialysis. ...
Stable oleuropein-coated gold nanoparticles in aqueous media were synthesized for the first time. Oleuropein (OLE) concentration in the reaction medium was found to greatly influence the outcome and stability of the resulting nanocolloid, with a marked decrease in particle size being found for the more concentrated oleuropein solutions. The protection mechanisms involved in the stabilized nanosystems were analyzed. Oleuropein self-assembled structures were found to be formed at a concentration threshold of [OLE] > 5 × 10−5 M, and observed through the use of CryoSEM imaging. Those structures were responsible for both the increased stability and the decrease in size observed at the more concentrated solutions.
... An alternative is to freeze the sample at high pressure, an idea first postulated by Moor and Riehle in 1968 [7]. At higher pressures, water expands less during freezing; hence, less heat of crystallization is produced, so adequate cryo-fixation is achieved at reduced cooling rates [8,9]. ...
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.
... High-pressure freezing (HPF) followed by freeze-substitution preserves the synaptic ultrastructure significantly better and avoids distortions of the cellular morphology induced by slow chemical fixation (Studer et al., 2001;Rostaing et al., 2006). In addition, it allows rapid vitrification within a few tens of milliseconds even for thicker specimens. ...
The presynaptic compartment of the chemical synapse is a small, yet extremely complex structure. Considering its size, most methods of optical microscopy are not able to resolve its nanoarchitecture and dynamics. Thus, its ultrastructure could only be studied by electron microscopy. In the last decade, new methods of optical superresolution microscopy have emerged allowing the study of cellular structures and processes at the nanometer scale. While this is a welcome addition to the experimental arsenal, it has necessitated careful analysis and interpretation to ensure the data obtained remains artifact-free. In this article we review the application of nanoscopic techniques to the study of the synapse and the progress made over the last decade with a particular focus on the presynapse. We find to our surprise that progress has been limited, calling for imaging techniques and probes that allow dense labeling, multiplexing, longer imaging times, higher temporal resolution, while at least maintaining the spatial resolution achieved thus far.
... This did not lead to freezing of the algae cells themselves but only to freezing of the surrounding aqueous medium. The further preparation in principle followed the instructions of the manufacturer as described by Studer [83]. First, the flat specimen carrier with the frozen alga suspension was mounted to the specimen pod. ...
Background: Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a deined frozen state during the entire ixation procedure is an essential prerequisite. However, current techniques are not able to ix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical ixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-ixation methods, such as high pressure freeze ixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented.
Results: Diferent algae (Micrasterias denticulata, Klebsormidium crenulatum) and higher plant tissues (Lemna sp., Ranunculus glacialis, Pinus mugo) were successfully frozen and prepared for HPF at freezing temperatures (− 2 °C, − 5 °C, − 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-ixation were performed without intermediate thawing. After cryosubstitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the diferent organisms when compared to specimens ixed at ambient temperature.
Conclusions: The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryofixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identiication of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.
... The use of cryo preparation methods aims at a near native preservation of cell structure. Ideally, the water content of the sample is vitrified after high-pressure freezing (Dahl & Staehelin, 1989;Moor & Riehle, 1968;Studer, Graber, Al-Amoudi, & Eggli, 2001). During freeze substitution the water in the sample is consecutively exchanged by organic solvents at −80 to −90 C to prevent formation of ice crystals (Steinbrecht & Müller, 1987). ...
For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.
... Dispersion was achieved by sonication for 24 h at 45°C to achieve dispersion. A relatively dilute suspension was prepared to reduce the effect of water crystallization in thick samples (>50 mm) [49]. The suspensions were placed in stub holes and plunge-frozen in liquid nitrogen (À200°C). ...
Clay mineral properties, together with solution chemistry, control the assembly of clay platelets into hierarchical structures, including tactoids and aggregates. We studied the effect of salinity on the assembly of kaolinite, illite, and montmorillonite at three critical scales: platelet, tactoid, and aggregate, using cryogenic scanning electron microscopy (cryo-SEM), atomic force microscopy (AFM) and cryo-transmission EM (cryo-TEM), respectively. Cyro-SEM images coupled with original alignment analysis indicate that the degree of aggregate alignment in an ionized solution was significantly higher than in deionized water. Furthermore, upon increasing platelet-platelet bonding energy (montmorillonite > illite > kaolinite), tactoid size increased, packing was less ordered, and aggregate alignment decreased. AFM measurements showed that an increase in ionic-strength caused a decrease in the Young's modulus of the clays, indicating higher tactoid alignment, since, disordered structures, comprising various platelet orientations, are stiffer than highly-aligned structures. We successfully measured distances <1 nm, for both kaolinite and montmorillonite by cryo-TEM, directly demonstrating that increasing ionic-strength reduces platelet-platelet distances. The outcome of this study offers a new approach and methodology to study fundamental colloid-assembly which will trigger future studies investigating additional parameters affecting assembly such as, temperature, solution pH, natural organic matter, and anthropogenic activity.
... Cells were grown using the methods described above and harvested at mid-log phase. Samples for TEM imaging were prepared as described before (40,42). In brief, bacteria were pelleted by centrifugation (5000 rpm for 5 minutes) and the supernatant was discarded. ...
The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown in fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-Sequencing revealed carbon source-dependent regulation of distinct genes of wildtype strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose-1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is < 10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.
... Apppareil de cryofixation sous haute pression EMPACT1, Leica(Studer et al., 2001) A-Ecran de contrôle, B-Réservoir pour le liquide de haute pression (Méthylcyclohexane), C-Zone de chargement du porte-échantillon et de transfert dans l'azote liquide après la cryofixation, D-Deware contenant la réserve d'azote liquide.II-2-3-Cryosubstitution et enrésinementAprès congélation sous haute pression, les échantillons sont transférés dans l'automate de substitution AFS (Freeze-substitution-Leica Vienne) (Figure 55) pré-refroidi à -140°C. Les échantillons sont substitués dans l'acétone anhydre contenant 0,5% d'acétate d'uranyle à -90°C pendant 72h. ...
La paroi primaire des plantes des dicotylédones vraies est constituée de microfibrilles de cellulose, de protéines et de polysaccharides complexes, représentés par les hémicelluloses et les pectines. Chez la plante modèle Arabidopsis thaliana, le xyloglucane (XyG) et le rhamnogalacturonane-I (RG-I) constituent les catégories majeures des hémicelluloses et des pectines respectivement. Avant d’être transportés vers la paroi, le XyG et le RG-I sont synthétisés au sein de l’appareil de Golgi, notamment via l’action d’enzymes d’interconversion des sucres et de glycosyltransférases, qui vont générer et assembler les différents sucres pour former les polysaccharides. L’objectif de ce projet de recherche a été d’effectuer la cartographie golgienne d’épitopes polysaccharidiques et de différentes enzymes impliquées dans la synthèse du XyG et du RG-I, ainsi que de rechercher l’existence de complexes enzymatiques, en utilisant les techniques de microscopie confocale à balayage laser, de microscopie électronique à transmission et d’immunocytochimie quantitative.
... The currently available equipment freezes samples at about 200,000 kPa. At this pressure, samples of up to 200 μm can be frozen without formation of ice crystals (108,(114)(115)(116). The combination of these two methods is now considered to be the best approach for ultrastructural studies of bacteria when no access to CET is possible. ...
The Gram-Positive Bacterial Cell Wall, Page 1 of 2
Abstract
The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.
... TEM images give the qualitative information about the microstructure of the samples. The samples were highpressure frozen with a Leica EM Pact machine (Studer et al., 2001), embedded in EPON epoxy resin, sliced with a microtome and imaged in the FEI Tecnai 12 microscope at working voltage of 120 kV. AFM imaging was used to calculate particle size and shape distribution of the clay particles. ...
The planned final disposal repositories of spent nuclear fuel in several countries, including Finland, pose significant scientific challenges due to their extremely long lifespan. One of the key materials proposed for use in Posiva Oy's repository in Finland is MX-80 bentonite in a compacted, water-saturated state. Border cases of calcium and sodium forms of purified bentonite were included in this study. The MX-80 in the repository is expected to undergo cation exchange due to the composition of the groundwater. The clays were studied at different dry densities between 0.7 and 1.6 g cm –3 . The microstructure of the water-saturated, compacted clays was investigated using small-angle X-ray scattering, nuclear magnetic resonance and transmission electron microscopy. Additionally, atomic force microscopy was used to characterize the shape and size of the fine-fraction clay platelets. As expected, the average shape of the fine fractions was smaller than the bulk material, but a more elongated shape was present in the purified material. Mainly due to sample density, the pore structure was noticeably different for the Na form of purified bentonite at 0.7 g cm –3 density, but at higher degrees of compaction, no significant differences were noted between the samples. The laboratory results obtained in this study could be useful for safety and performance analysis of this high-level waste repository where sodium bentonite is used and is expected to change its charge-compensating cation composition during the repository's lifetime. Microstructural data may be used in modelling of diffusion and sorption by surface complexation modelling, for example, or as a basis for mechanical and water transport models.
... On the other hand, highpressure freezing (HPF) and other physical fixation approaches (e.g., slam freezing) are preferable to chemical fixation as they allow fixation within milliseconds, rather than seconds to minutes for chemical fixation. But they pose their own set of limitations (Studer et al. 2001). Only small tissue samples of about 300 μm in thickness can be frozen without ice crystal damage (McDonald and Auer 2006), which means that tissues like the retina need to be exposed by ocular enucleation and dissection prior to HPF. ...
The phototransductive membrane disks of a vertebrate photoreceptor outer segment (OS) are highly susceptible to perturbations during preservation for electron microscopy. To optimize their preservation for nanostructural studies, such as with electron tomography (ET), we developed a protocol, using a combination of chemical and physical fixation approaches, including transcardiac perfusion, high-pressure freezing, and freeze-substitution. © 2018, Springer International Publishing AG, part of Springer Nature.
... On the other hand, highpressure freezing (HPF) and other physical fixation approaches (e.g., slam freezing) are preferable to chemical fixation as they allow fixation within milliseconds, rather than seconds to minutes for chemical fixation. But they pose their own set of limitations (Studer et al. 2001). Only small tissue samples of about 300 μm in thickness can be frozen without ice crystal damage (McDonald and Auer 2006), which means that tissues like the retina need to be exposed by ocular enucleation and dissection prior to HPF. ...
Purpose:
To identify the molecular basis of inherited retinal degeneration (IRD) in a familial case of Pakistani origin using whole-exome sequencing.
Methods:
A thorough ophthalmic examination was completed, and genomic DNA was extracted using standard protocols. Whole exome(s) were captured with Agilent V5 + UTRs probes and sequenced on Illumina HiSeq genome analyzer. The exomeSuite software was used to filter variants, and the candidate causal variants were prioritized, examining their allele frequency and PolyPhen2, SIFT, and MutationTaster predictions. Sanger dideoxy sequencing was performed to confirm the segregation with disease phenotype and absence in ethnicity-matched control chromosomes.
Results:
Ophthalmic examination confirmed retinal degeneration in all affected individuals that segregated as an autosomal recessive trait in the family. Whole-exome sequencing identified two homozygous missense variants: c.1304G > A; p.Arg435Gln in ZNF408 (NM_024741) and c.902G > A; p.Gly301Asp in C1QTNF4 (NM_031909). Both variants segregated with the retinal phenotype in the PKRD320 and were absent in ethnically matched control chromosomes.
Conclusion:
Whole-exome sequencing coupled with bioinformatics analysis identified potential novel variants that might be responsible for IRD.
... The ash freezing process solidies the water and therefore prevents the molecular and ionic transportation. 154 Metabolic reactions cease, tissue structures are preserved, and the mechanical properties of the sample are strengthened allowing it to be properly sliced. Cryoxation can be performed by plunging the sample and cryogen or through high pressure freezing. ...
The ability to provide a fast and multielemental analytical response directly from a solid sample makes both laser-induced breakdown spectroscopy (LIBS) and X-ray fluorescence spectrometry (XRF) very versatile tools for plant nutrition diagnosis. This review focuses on the main developments and advances in LIBS and XRF in the analysis of plant materials over the last ten years. Fundamental aspects and instrumentation are given for both techniques. The developments in the quantitative analysis of plant leaves are discussed, with special emphasis on the key aspects and challenges concerning field sampling protocols, sample preparation, and calibration strategies. Microchemical imaging applications by LIBS and XRF (including synchrotron radiation) are also presented in a broader selection of plant compartments (e.g., leaves, roots, stems, and seeds). Challenges, expectations and complementarities of LIBS and XRF towards plant nutrition diagnosis are thoroughly discussed.
... The most widely used method is high-pressure freezing (HPF) [27]: after a few milliseconds during which pressure is raised to ∼2000 bar, rapid cooling proceeds. In this manner, samples up to ∼100-300 m thick can be vitrified [28,29]. Subsequently, vitreous sections (∼25-100 nm thick) have to be cut with a cryo-ultramicrotome [30,31]. ...
Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware. These developments also greatly increased the usability and applicability of CET as a diagnostic and research tool, which is now enabling structural biologists to determine the structure of proteins in their native cellular environment to sub-nanometer resolution. These recent technical developments have stimulated us to update on our previous review (Koning, R.I., Koster, A.J., 2009. Cryo-electron tomography in biology and medicine. Ann Anat 191, 427-445) in which we described the fundamentals of CET. In this follow-up, we extend this basic description in order to explain the aforementioned recent advances, and describe related 3D techniques that can be applied to the anatomy of biological systems that are relevant for medicine.
Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.
Type 2 diabetes (T2D) affects more than 32.3 million individuals in the United States, creating an economic burden of nearly $966 billion in 2021. T2D results from a combination of insulin resistance and inadequate insulin secretion from the pancreatic β cell. However, genetic and physiologic data indicate that defects in β cell function are the chief determinant of whether an individual with insulin resistance will progress to a diagnosis of T2D. The subcellular organelles of the insulin secretory pathway, including the endoplasmic reticulum, Golgi apparatus, and secretory granules, play a critical role in maintaining the heavy biosynthetic burden of insulin production, processing, and secretion. In addition, the mitochondria enable the process of insulin release by integrating the metabolism of nutrients into energy output. Advanced imaging techniques are needed to determine how changes in the structure and composition of these organelles contribute to the loss of insulin secretory capacity in the β cell during T2D. Several microscopy techniques, including electron microscopy, fluorescence microscopy, and soft X‐ray tomography, have been utilized to investigate the structure‐function relationship within the β cell. In this overview article, we will detail the methodology, strengths, and weaknesses of each approach. © 2024 American Physiological Society. Compr Physiol 14:5243‐5267, 2024.
This chapter is focused on imaging of viruses and viral vectors individually or in the context of cells and tissue. Common preparation techniques performed at room temperature or in cryogenic conditions for electron microscopy are summarized. Additional topics about instrumentation, data processing, correlative light and electron microscopy, selected applications, and future perspectives are included.KeywordsTransmission electron microscopy (TEM)Cryogenic transmission electron microscopy (cryoTEM)Correlative light and electron microscopy (CLEM)TomographyHigh-pressure freezing (HPF)Plunge freezingFreeze substitution (FS)Negative staining (NS)
Gas hydrates (clathrate hydrates, clathrates, or hydrates) are crystalline inclusion compounds composed of water and gas molecules. Methane hydrates, the most well-known gas hydrates, are considered a menace in flow assurance. However, they have also been hailed as an alternative energy resource because of their high methane storage capacity. Since the formation of gas hydrates generally require extreme conditions, developing porous material hosts to synthesize gas hydrates with less-demanding constraints is a topic of great interest to the materials and energy science communities. Though reports of modeling and experimental analysis of bulk gas hydrates are plentiful in the literature, reliable phase data for gas hydrates within confined spaces of nanoporous media has been sporadic. This review examines recent studies of both experiments and theoretical modeling of gas hydrates within four categories of nanoporous material hosts that include porous carbons, metal-organic frameworks, graphene nanoslits, and carbon nanotubes. We identify challenges associated with these porous systems and discuss the prospects of gas hydrates in confined space for potential applications.
With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information—known as multimodal imaging or correlative microscopy (CM)—has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution.
Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artifacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM.
To minimize the delay separating live imaging from cryo-arrest, we developed a high-pressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.
Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in December 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here we provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. We will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.
Viruses are obligatory intracellular parasites that reprogram host cells upon infection to produce viral progeny. Here, we review recent structural insights into virus-host interactions in bacteria, archaea, and eukaryotes unveiled by cellular electron cryo-tomography (cryoET). This advanced three-dimensional imaging technique of vitreous samples in near-native state has matured over the past two decades and proven powerful in revealing molecular mechanisms underlying viral replication. Initial studies were restricted to cell peripheries and typically focused on early infection steps, analyzing surface proteins and viral entry. Recent developments including cryo-thinning techniques, phase-plate imaging, and correlative approaches have been instrumental in also targeting rare events inside infected cells. When combined with advances in dedicated image analyses and processing methods, details of virus assembly and egress at (sub)nanometer resolution were uncovered. Altogether, we provide a historical and technical perspective and discuss future directions and impacts of cryoET for integrative structural cell biology analyses of viruses.
Expected final online publication date for the Annual Review of Virology, Volume 7 is September 29, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Cryofixation by high pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of < 200 μm thickness and about 2000 μm across. A wool follicle is a rare intact organ in a single sample about 200 μm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes during wool fibre development, limited water movement and accessibility of fixatives are some other issues negatively affecting the preservation of the follicle ultrastructure via conventional chemical processing. Here we show for the first time that HPF‐FS of wool follicle could yield high quality tissue preservation for ultrastructural studies of wool follicles using transmission electron microscopy. This article is protected by copyright. All rights reserved
Cryomethods for Biological EM Specimen Preparation - Volume 9 Issue S02 - Kent L. McDonald
The skin is the largest organ of the body. It is a natural barrier between the environment and the organism that plays a key role in the protection of the body against abiotic or biotic stress. The skin is made up of three different cell layers, namely the hypodermis, the dermis and the epidermis. The epidermis is, in turn, composed of four different layers, the basal, the spinous, the granular and the horny layer. In the epidermis, keratinocytes is the major cell type known to originate from basal cells. In the present work, we have studied the expression of a number of genes (antimicrobial peptide genes and pro-inflammatory cytokine genes) in different layers of healthy human epidermis using quantitative real-time PCR coupled to Laser Capture Microdissection (LCM). Using the same approach, we have also investigated the effect of bacteria belonging the human epidermal microbiota on the expression of the same genes within the epidermal layers. In addition, the spatial distribution of gene products has also been examined using immunocytochemsitry. The major findings are: 1) We have developed an optimized LCM protocol for the isolation of human epidermal layers. We have been able to isolate two fractions highly enriched in keratinocytes from the basal and the granular layers (Percoco et al. 2012. Experimental Dermatology. 21, 531-534). Such a method can now be easily applied in dermatological research. 2) We have found that, in healthy human epidermis, innate immunity-related genes including human beta-defensin genes are differentially expressed within different layers. 3) We have found that the three bacteria Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas fluorescens induce a significant increase in the expression of beta-defensin (hBD2 and hBD3) and pro-inflammatory cytokines (IL-1 alpha, IL-1 beta and IL-6) genes. 4) We have localized the gene products within different cell layers using specific antibodies in conjunction with immunofluorescence and immunogold-electron microscopy (Percoco et al., Submitted).
This chapter describes how different preparation techniques for cryo‐scanning electron microscope (cryo‐SEM) can be combined, how these individual steps are implemented on instruments available from Leica Microsystems, and how the sample can be protected between these steps. One major subject of the chapter is the Leica EM VCT500 vacuum cryo‐transfer system linking preparation instruments both to each other and also to the SEM. Unless the surfaces to be studied in cryo‐SEM are naturally exposed, bulk frozen hydrated specimens have to be mechanically opened up to make their inner part accessible for analysis: one approach is the freeze fracture (FF) technique, where parts of the specimen are cleaved under high vacuum. Both the secondary electron (SE) as well as the backscattered electron (BSE) detectors can be used for signal generation in cryo‐SEM. The VCT cryo‐SEM set available from Leica Microsystems is implemented identically for the VCT100 and VCT500 vacuum cryo‐transfer systems.
Predating the cryo‐field emission gun scanning electron microscopy (cryo‐FEGSEM), and for that matter cryo‐electron microscopy (EM), was the use of low temperature to preserve the viability of biological tissues. This chapter describes the states of water at low temperatures, damage related to ice nucleation and growth, and strategies to avoid such damage. Freeze drying of a frozen biological sample involves the sublimation of ice, and the simultaneous and/or subsequent removal of some or all of the water that was not converted to ice during the initial freezing. Cryoprotectants, which are essential for minimising cryoinjury during freezing, may be toxic to biological systems. Monohydric alcohols, dimethyl sulfoxide (DMSO) and ethylene glycol (EG) are known to denature enzymes at room temperature. Vitrification allows for structures to be viewed in their native state, avoiding the artefacts commonly associated with chemical fixation and dehydration preparation techniques as well as the ultrastructural disruption attributed to intracellular ice formation.
A site-specific, cryogenic, focused ion beam (FIB) method is presented for the preparation of atom probe tomography (APT) specimens from a frozen liquid/solid interface. As a practical example, the interface between water and a corroded boroaluminosilicate glass has been characterized by APT for the first time. The water/glass interface is preserved throughout specimen preparation by plunge freezing the corroding glass particles with the corrosion solution into slush nitrogen. Site-specific specimen preparation is enabled through a new approach to extract and mount a small volume of material using a cryogenically cooled FIB stage and micromanipulator. The prepared APT specimens are subsequently transferred from the FIB to APT under cryogenic and high-vacuum conditions using a novel FIB/APT transfer shuttle and home-built environmental transfer hub attached to the APT system. Particular focus is given to the technical methods for specimen fabrication under cryogenic conditions. Persistent challenges are discussed in addition to future opportunities for this new specimen preparation method.
There have been great efforts on the nanoscale 3D probing of brain tissues to image subcellular morphologies. However, limitations in terms of tissue coverage, anisotropic resolution, stain dependence, and complex sample preparation all hinder achieving a better understanding of the human brain functioning in the subcellular context. Herein, X‐ray nanoholotomography is introduced as an emerging synchrotron radiation‐based technology for large‐scale, label‐free, direct imaging with isotropic voxel sizes down to 25 nm, exhibiting a spatial resolution down to 88 nm. The procedure is nondestructive as it does not require physical slicing. Hence, it allows subsequent imaging by complementary techniques, including histology. The feasibility of this 3D imaging approach is demonstrated on human cerebellum and neocortex specimens derived from paraffin‐embedded tissue blocks. The obtained results are compared to hematoxylin and eosin stained histological sections and showcase the ability for rapid hierarchical neuroimaging and automatic rebuilding of the neuronal architecture at the level of a single cell nucleolus. The findings indicate that nanoholotomography can complement microscopy not only by large isotropic volumetric data but also by morphological details on the sub‐100 nm level, addressing many of the present challenges in brain tissue characterization and probably becoming an important tool in nanoanatomy.
La sélénoprotéine T (SelT) est une protéine thiorédoxine-like abondamment exprimée au cours du développement embryonnaire chez le rat, mais son expression tend à disparaître après la naissance, notamment dans le coeur, suggérant un rôle limité de la SelT à l’âge adulte. Néanmoins, nous avons pu montrer que la SelT est réexprimée au niveau cardiaque suite à une ligature de l’artère coronaire (LC), suggérant le rôle potentiellement protecteur de cette protéine au cours des pathologies cardiovasculaires. Le but de notre projet fut donc d’évaluer les effets cardiaques d’une thérapie par la SelT au cours de l’insuffisance cardiaque, moyennant soit une thérapie protéique, soit une thérapie génique visant à surexprimer la SelT au niveau cardiaque ou au niveau systémique. La supplémentation en SelT (15μg/kg/jour, minipompes ip) a permis d’améliorer significativement le débit cardiaque et la fraction de raccourcissement du VG, mais également d’améliorer les pressions télé-systoliques et télé-diastoliques du ventricule gauche ainsi que la perfusion coronaire. Ces changements sont associés à une diminution du stress oxydant cardiaque ainsi qu’à une répression des mécanismes inflammatoires cardiaques. L’ensemble de ces améliorations a été observé sans modification de la taille d’infarctus. En parallèle, nous avons pu montrer qu’une injection intraveineuse d’un rAAV9-SelT (1.1011vg) une semaine après la LC permettait de diminuer significativement la dilatation ventriculaire gauche 3 mois après la LC. De manière concomitante, la thérapie génique par la SelT améliore le débit cardiaque ainsi que la perfusion cardiaque. Ces changements sont associés à une amélioration de la compliance et de l’élastance cardiaque. Par ailleurs, l’injection intramusculaire d’un rAAV8-SelT suivant le même protocole que précédemment. Nous avons pu montrer que le traitement par cet AAV permettait de diminuer significativement la dilatation du VG et d’améliorer la fraction de raccourcissement. De plus, la thérapie génique a permis d’améliorer la perfusion cardiaque ainsi que la relaxation coronaire endothélium-dépendante. Nous avons également pu montrer que l’ensemble des effets de la SelT sont médiés par le résidu Sec, dès lors que la modification de ce résidu par une alanine, annihile totalement l’ensemble des effets positifs observés au cours de notre étude. Ainsi, nos résultats ont permis de montrer clairement que le rôle bénéfique d’un traitement par la SelT au cours de l’ICC, et ce, grâce à un mécanisme sélénocystéine-dépendant. La SelT semble donc être une cible thérapeutique prometteuse pour le traitement de cette pathologie.
Plasmodesmata (PD) are nanometric (similar to 20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from cell-to-cell and then systematically throughout the whole plant. Their central position in plant biology makes it crucial to understand their physiology and especially link their function to their structure. Over the past 50 years, electron microscopists have observed them and attempted to ultrastructurally characterize them. They laid the foundation of what is known about these pores (Tilney et al., 1991; Ding et al., 1992; Oparka and Roberts, 2001; Nicolas et al., 2017a). Despite the explosion of three-dimensional electron microscopy (3D-EM), PD ultrastructure remained recalcitrant to such technique. The first technical difficulty is to process them in such a way where they are as close to their native state as possible. Secondly, plant samples reveal themselves as being difficult to process due to the poor staining/fixating reagents penetration rates, their increased size, their high water content and the presence of an acidic vacuole. On top of this, their very unique position in the cell wall and their nanometric size make them difficult to conveniently stain in order to see the inner-workings of these pores. Here we describe in detail the protocol used in Nicolas et al. (2017b) to image PD in fine detail and produce high-resolution tomograms.
Bacterium-host interactions are important for diverse ecological settings including pathogenicity and symbiosis. Electron cryotomography is a powerful method for studying the macromolecular complexes that mediate such interactions in situ. The main limitation of electron cryotomography is its restriction to relatively thin samples such as individual bacterial cells. Cryo-focused ion beam milling was recently proposed as a solution to the thickness limitation. This approach allows the artifact-free thinning of biological specimens for subsequent imaging in the transmission electron microscope. By enabling near-native imaging of bacteria inside their eukaryotic host, this combination of techniques promotes the integration of data from structural biology and infection biology. Therefore, electron cryotomography associated with cryo-focused ion beam milling holds great potential for establishing multiscale models of cell-cell interactions from the atomic, to the cellular and to the intercellular scale.
Determining the in situ localization of trace elements at high lateral resolution levels in the biological system is very challenging, but critical for our understanding of metal sequestrationa and detoxification. Here, the cellular and subcellular distributions of Cu and Zn in contaminated oysters of Crassostrea hongkongensis were for the first time mapped using nanoscale secondary ion mass spectrometry (nanoSIMS). Three types of metal-containing cells were revealed in the gill and mantle of oysters, including Cu-specific hemocytes, Cu and Zn-containing granular hemocytes, and Cu and Zn-containing calcium cells. Obvious intercellular distribution of Cu was found in the gill tissue, indicating the potential role of hemolymph in the transportation of Cu in oysters. The distribution of Cu showed a strong co-localization with sulfur and nitrogen in Cu-specific hemocyte and intercellular hemolymph. In the Cu and Zn-containing granular hemocytes and calcium cells, the co-occurrence of Cu and Zn with phosphorous and calcium was also found. Different relationships of distributions between Cu/Zn and macronutrient elements (nitrogen, sulfur and phosphorous) implied the differential metal complexation in oysters. Interestingly, quantitative analysis of the ratios of 32S–/12C14N– and 31P–/12C14N– of metal-deposited sites suggested the dynamic process of transfer of Cu and Zn from the metabolized protein pool to a more thermodynamically stable and detoxified form.
To date, high pressure freezing is the only reliable cryofixation method for vitrifying bulk biological samples up to 150μm in thickness (1). This proves the enormous potential of the method. Nevertheless, the procedure is not well accepted by the scientific community. It seems that people not completely convinced by the method have reservations to use high pressure freezing machines. The main reasons seem to be that the commercially available machines are noisy, large, very heavy, expensive and consume a lot of liquid nitrogen. Last but not least, a relatively large volume of cryogen is pressurized to 2000bar representing a potential risk of accident. Considering high pressure freezing to be a very potent method, it was tried to establish a new machine avoiding the above mentioned disadvantages.
The first functional high pressure freezing apparatus was freezing fine metal rods with a boring of 0.2mm in diameter (2). This boring was filled with suspensions, the rod was connected to a pressure generator thus pressurizing the suspension, and simultaneously the metal rod was frozen from the outside by a low pressure jet of liquid nitrogen. The big disadvantage of the method was that only freeze-fracture experiments could be performed at the time because the sample could not be removed from the boring. As a consequence, this approach was never commercialised. In the mean time sample preparation has improved. Cellulose capillaries can be used to freeze suspensions and tissue samples (3,4). To freeze these capillaries in the new machine, they are introduced in a sample holder which consists of a copper tube with an inner diameter of 0.3mm and a length of 16mm. This tube fits into a concentrical stainless steel tube which stabilises the copper rod and helps to fix the sample in the cryofixation device.
Pressure-freezing has often been regarded as a method for highly technical specialists. At the beginning of its development, this may have been true: it was introduced in 1968 by Moor and Riehle at the European conference on electron microscopy in Rome. The interest of the audience was not overwhelming, because everybody thought that this approach is oversophisticated and in principle unnecessary. In the following decade, many technically less pretentious freezing methods have been developed, which work in the absence of pressure. All of them became standardized and their methodology has been described in numerous reviews and textbooks (e.g. Rash 1983; Gilkey and Staehlin 1986; see also Sitte et al., Chap. 4, this Vol.). The compiled experience shows the manifold profits of applying impact-, plunge-, jet- and spray-freezing. In one aspect, however, all of these techniques are inadequate: namely they only enable satisfactory cryofixation of objects or superficial layers, which are not thicker than 10–20 μm. This limitation is caused by the physical properties of aqueous systems and it indicates that thicker specimens can be well cryofixed only if these properties are altered.
We have developed three instruments to freeze different biological specimens for various purposes in the easiest and most economical way: a punching device, a rapid immersion device and a device to freeze the specimens against a copper block cooled by liquid helium. Cooling rate measurements are made by a thin flat thermocouple. Freezing against a copper block cooled down to 4 K gives cooling rates 3–5 times higher than freezing by immersion into propane at 85 K.
Biological specimens prepared for cryoelectron microscopy seem to suffer less damage when they are frozen under 2 kbar pressure rather than under normal conditions. The volume that can be well preserved is larger. This fact has been illustrated in a number of publications on a number of different samples. However, there is a lack of quantitative data concerning the depth of this good specimen preservation.
Catalase crystals in various sugar solutions have been used as test objects and vitrification, as determined by electron diffraction, has been used as the criterion for good freezing. Keeping all other conditions equal, the depth of vitrification is approximately 10 times larger with freezing at high, rather than normal, pressure. The high-pressure vitrification depth in a 15–20% sugar solution averages 200 μm. Fully vitrified specimens up to 700 μm in thickness are obtained. When crystalline water is observed it is frequently in the form of high-density ice II, III or IX.
These results are probably also relevant for typical biological specimens. The advantage of high-pressure freezing must be balanced by the possible consequences of a considerably increased cooling time and by the damage that may be induced by the pressure.
High-pressure freezing of chemically untreated nodules of soybean (Glycine max (L.) Merr.), in sharp contrast to chemical fixation and prefixation, appears to preserve the ultrastructure close to the native state. This is supported by the observation that the peribacteroid membrane of high-pressure-frozen samples is tightly wrapped around the bacteroids, a finding that is fully consistent with the current views on the physiology of oxygen and metabolite transport between plant cytosol and bacteroids. In soybean root nodules, the plant tissue and the enclosed bacteria are so dissimilar that conventional aldehyde-fixation procedures are unable to preserve the overall native ultrastructure. This was demonstrated by high-pressure freezing of nodules that had been pre-fixed in glutaraldehyde at various buffer molalities: no buffer strength tested preserved all ultrastructural aspects that could be seen after high-pressure freezing of chemically untreated nodules.
Electron probe microanalysis data on the intracellular content and distribution of electrolyte ions depends critically on the functional state of the cells at the moment of cryofixation. Whereas tissue specimens often require special in-situ freezing techniques, isolated and cultured cells can be frozen within their environmental medium under physiologically controlled conditions. Thus, they represent a feasible system to study functional ion-related intracellular parameters such as the K/Na ratio. Specifically modified freezing devices allow the study of ion shifts related to dynamic processes in cells, for example, locomotion and exocytosis. The time resolution achieved by time-controlled cryofixation is approximately 1 ms.
High pressure freezing permits the successful cryoimmobilization of thick biological specimens (up to approx. 500 microns). A very high yield of adequately frozen specimens, in which no segregation patterns due to ice crystal formation is apparent after freeze-substitution or freeze-fracturing, is obtained with suspensions of microorganisms as well as plant and animal tissue. This very high yield is attributed to an optimized transfer of pressure and cold to the biological specimen. This is achieved by replacement of extraspecimen water or buffer by 1-hexadecene, a chemically inert, hydrophobic paraffin oil of low viscosity and low surface tension.
The recognition that the endoplasmic reticulum (ER), rather than the mitochondria, is the main organelle regulating the cytoplasmic Ca2+ concentration in non-muscle cells supports the notion that an alternative physiological role of mitochondrial Ca transport is the modulation of Ca-sensitive mitochondrial enzymes through small (micromolar) fluctuations in the concentration of mitochondrial matrix Ca2+. The latter mechanism could operate only if the mitochondrial Ca concentration were low, as it is in muscle and retinal rods, below the levels saturating the regulated enzymes. In contrast, if the ER serves as an intracellular Ca store, its Ca content would be expected to be high. In view of the major metabolic function of the liver, the question of whether hepatic mitochondrial matrix Ca2+ regulates metabolism is particularly important, but the range of Ca concentrations reported for isolated liver mitochondria is too wide to provide a conclusive answer. Therefore, we have used electron probe X-ray microanalysis (EPMA) to measure the subcellular distribution of Ca in liver snap-frozen in vivo, and report here that the endoplasmic reticulum is a major intracellular store of Ca, while the concentration of Ca in mitochondria is low and compatible with the regulation of mitochondrial enzymes.
For more than 20 years, high-pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high-pressure freezing. The cooling rates necessary to achieve this result under high-pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed.
A procedure for efficient cryoimmobilization of large volumes of cell suspensions or micro-organisms by high-pressure freezing is described. This procedure uses transparent, porous cellulose capillary tubes with an inner diameter of 200 μm, into which the suspensions are drawn by capillary action. The tubes are processed by high-pressure freezing and freeze-substitution as if they were tissue samples. Centrifugation of suspensions at low temperatures is no longer necessary and cryopreparation is greatly facilitated.
A very high yield of adequately frozen specimens is obtained due to the constant, defined sample geometry. This approach can also be used to process suspensions by conventional chemical fixation, eliminating the need to embed pellets in low-melting-point agarose, for example, prior to chemical fixation.
The preparation procedure is demonstrated with suspensions of nematodes, paramecia and bacteria.
Mixtures of ethanol, 2-propanol and 2-butanol can be used as a cryoglue to mount vitrified biological specimens for ultrathin cryosectioning. Brought directly from room temperature to a cutting support at 140 K in the cold chamber of a cryoultramicrotome, these alcohols stiffen to a viscous and gluey consistency allowing the attachment or embedding of a vitreous biological sample. The mass hardens at lower temperatures fixing the sample well for microtomy. With ethanol: 2-propanol (2:3), samples are applied at 140 K and ultrathin cutting can be done at 115 K.
High-pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze-substitution) of biological tissue up to a thickness of about 200 microns. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine-needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s of excision.
High-pressure freezing (HPF) is currently the only method which enables adequate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and preparation methods, the technique has not yet reached its full potential. While suspensions can be prepared easily for HPF, tissue preparation is restricted by the need to compromise between different requirements and difficulties. (i) In order to achieve optimal freezing quality, very thin samples are required. (ii) There is mechanical difficulty in cutting such thin samples without distorting the organization of the tissue. (iii) The cutting and the succeeding preparation steps of small samples require long handling times (minutes), which may result in physiological and hence structural alterations. Computerized heat transfer simulations are presented which confirm that the efficiency of heat extraction from cylindrical samples contained within thin-walled metal tubes is higher than from standard flat discoid samples sandwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the quick excision of such cylinders of soft tissues. The device utilizes sharp gold needles of an inner diameter of 200 microm and wall thickness of 50 microm. The frozen sample contained in the soft gold needle permits all the manipulations needed for conventional cryo-preparation techniques for electron microscopy (e.g. cryo-sectioning, freeze-fracturing, freeze-substitution).
A method for rapid freezing is described in which use is made of the good heat conducting properties of silver. The freezing was accomplished by bringing the tissue in contact with a polished silver surface at the temperature of liquid nitrogen either at atmospheric or reduced pressure. Helium gas flowing over this surface prevented the condensation of water or air on the silver. After freezing the tissue was placed in a substituting solvent. The best results were obtained with 2% osmium tetroxide in acetone at −85°C. The ultrastructure of the tissue was well preserved in a narrow surface layer only.
We also thank all collaborators of Leica Microsystems (Vienna) involved in this project for support. The first author is grateful for being generously supported by
- Anatomy
Anatomy) for technical assistance. We also thank all collaborators of Leica Microsystems (Vienna) involved in this project for support. The first author is grateful for being generously supported by Professor J. Dubochet (LAU, Universite  de Lausanne) and Professor E. B. Hunziker (M.E. Mu È ller Institute of Biomechanics, University of Bern).
Ueber die Vitrifizierung verdünnter wässeriger Lösungen
- U Riehle