High-pressure Freezer Research Articles

Chloroplast-actin filaments decide the direction of chloroplast avoidance movement under strong light in Arabidopsis thaliana.

Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.

Cryo-FIB preparation of whole cells and tissue for cryo-TEM: use of high-pressure frozen specimens in tubes and planchets.

SummaryThe desire to study macromolecular complexes within their cellular context requires the ability to produce thin samples suitable for cryo‐TEM (cryo‐transmission electron microscope) investigations. In this paper, we discuss two similar approaches, which were developed independently in Utrecht (the Netherlands) and Albany (USA). The methods are particularly suitable for both tissue samples and cell suspensions prepared by a high‐pressure freezer (HPF). The workflows are explained with particular attention to potential pitfalls, while underlying principles are highlighted (‘why to do so’). Although both workflows function with a high success rate, full execution requires considerable experience and remains demanding. In addition, throughput is low. We hope to encourage other research groups worldwide to take on the challenge of improving the HPF– cryo‐FIB‐SEM – cryo‐TEM workflow. We discuss a number of suggestions to this end.Lay DescriptionLife is ultimately dictated by the interaction of molecules in our bodies. Highly complex equipment is being used and further developed to study these interactions. The present paper describes methods to prepare small, very thin lamellae (area of 5×5 µm2, thickness 50–300 nm) of a cell to be studied in a cryo‐transmission electron microscope (cryo‐TEM). Special care must be taken to preserve the natural state of molecules in their natural environment. In the case of cryo‐TEM, the samples must be frozen and kept frozen to be compatible with the vacuum conditions in the microscope. The frozen condition imposes technical challenges which are addressed. Two approaches to obtain the thin lamellae are described. Both make use of a focused ion beam (FIB) microscope. The FIB allows removal of material with nanometre precision by focusing a beam of ionised atoms (gallium ions) onto the sample. Careful control of the FIB allows cutting out of the required thin lamellae. In both strategies, the thin lamellae remain attached to the original sample, and the ensemble of sample with section and sample holder is transported from the FIB microscope to the TEM while being kept frozen.

Stages of the symbiotic zooxanthellae–host cell division and the dynamic role of coral nucleus in the partitioning process: a novel observation elucidated by electron microscopy

The relationship of the symbiotic algae or commonly referred as zooxanthellae (zo) and its host invertebrate species is uniquely observed among cnidarians where these tiny single-celled endosymbionts are used to reside densely within the gastrodermal tissue. However, at the ultrastructural level, little has been known on how zo are maintained through proliferation within its host cells. In the present study, the stages of division of the zo that occurred in the coral cells were investigated by electron microscopy. Scleractinian Pocillopora damicornis was fixed by freeze-substitution methods using a high-pressure freezer and embedded in resin. Ultrathin sections were observed using transmission electron microscope. Symbiotic cell at interphase showed the cortical fine structures of the non-dividing zo enclosed by the outermost plasma membrane (zpm). Overlying them, two distinct layers of intermediate membranes (inm) and a symbiosome membrane (sm) were found. When cytokinesis occurred, the zpm at the opposite division regions formed ‘U-shaped’ furrows which led amphiesmal vesicle (av) to protrude inwardly. As they invaginates, new avs and series of cortical microtubules (mi) are used to develop concomitant with the symbiont’ nuclear division. After the cytokinesis, the overlying membranes successively invaginate; thus, the daughter zooxanthellae were transformed into coccoid cells. These cells, however, remain contiguous to each other until when the coral nucleus (cnu) migrated toward one of the cleavage sites to intervene and dissociate them. Upon occupying the interzooxanthellar space, host nuclear division occurred prior to cytokinesis. These events involving the role of cnu in the partitioning process are first reported in this study, in which its dynamic intervening behavior had been elucidated as a novel finding from the cnidarian species.

In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

Thin polyester filters as versatile sample substrates for high-pressure freezing of bacterial biofilms, suspended microorganisms and adherent eukaryotic cells.

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

Nanostructural Evolution during Catalyst Layer Formation Studied via Cryo-Electron Microscopy

Polymer electrolyte fuel cells (PEFCs) have recently received considerable attention as a next-generation power source, since from the practical viewpoints of energy efficiency and environmental friendliness, they compare favorably with present energy sources1. The catalyst layer (CL) of membrane electrode assembly (MEA) in the PEFC consists from the carbon supported platinum (Pt) nanoparticle and ionomer. The CL is fabricated by coating of the catalyst ink containing the platinum-loaded carbon (Pt/C) catalyst, ionomer, and solvents. The electrochemical activity and transport phenomena in a MEA are affected by the nanostructures in the CL2,3. Though many research activities have been reported on a structural formation in the CL4,5, the dynamics of the CL formation process during the solvent evaporation is still unclear. In this study, we have demonstrated that the structural evolution in the catalyst inks and CLs in drying process was elucidated by cryogenic scanning electron microscopy (cryo-SEM) and cryogenic transmission electron microscopy (cryo-TEM). The catalyst ink was prepared by blending a Pt/C catalyst, ionomer (Nafion®) and solvents (water/1-propranol (NPA) =1:1 in volume ratio). The solid content of the catalyst ink was 10 wt.%, and the weight ratio of ionomer to carbon ratio was 1.0. The catalyst ink was frozen using a high-pressure freezer (EM ICE, Leica Microsystems, Vienna, Austria). The block surface of the frozen catalyst ink for the cryo-SEM observation was then trimmed with a cryo ultramicrotome (EM UC7/FC7T, Leica Microsystems, Vienna, Austria). The CLs were prepared by following procedure. The catalyst ink was coated on Cu substrates (3 mm diameter) placed on a PTFE film using a film applicator. The Cu substrates coated catalyst ink were picked up in 60 and 180 sec after the coating, and soaked into liquid ethane for freezing (CL60 and CL180, respectively). The dried CL was prepared by drying over an hour at room temperature (CLdry). The frozen catalyst ink and catalyst layers on the Cu substrates were observed with a scanning electron microscope (JSM-6701F, JEOL, Tokyo, Japan) incorporated with a liquid nitrogen cooled-stage (EM VCT100, Leica Microsystems, Vienna, Austria). The cryo-SEM image of the catalyst ink is shown in Fig. 1(a). The brighter particle and darker region correspond to the Pt catalyst nanoparticles and the frozen solvents, respectively. The Pt/C particles formed the several hundred nanometers to micrometer size agglomerates in the catalyst ink. The cryo-SEM images of the surface structure on the CL60 and CL180 are shown in Fig. 1(b) and (c), respectively. The bright particles with several-hundred nanometers correspond to the frost on the sample surface. In CL60, the shape of sample surface of the CL was flat and the agglomerates of Pt/C particles observed in the catalyst ink could not be seen on the surface. In CL180, the several ten micrometers agglomerates of the Pt/C particles could be seen in the CL. In contrast, the solvents and ionomers exhibited the network structure, which connected between the Pt/C agglomerates. In CLdry, the network structure does not exist any longer as shown in Fig. 1(d). In the CL formation process, the size of agglomerated of the Pt/C particles grew to larger agglomerates induced by solvent evaporation. The structure of the CL was changed the liquid–like to the solid via the network structure. The submicron structural evolution in the CL formation process during the solvent evaporation is demonstrated by the cryo-SEM observations. The further nanostructural investigations of the catalyst ink and the CL using cryo-TEM are now in progress. Acknowledgment This presentation is based on results obtained from the PEMFC Research and Development Program for “Highly‐Coupled Analysis of Phenomena in MEA and its Constituents and Evaluation of Cell Performance” commissioned by the New Energy and Industrial Technology Development Organization (NEDO).

Preparing Fission Yeast for Electron Microscopy.

Freezing samples while simultaneously subjecting them to a rapid increase in pressure, which inhibits ice crystal formation, is a reliable method for cryofixing fission yeast. The procedure consists simply of harvesting cells and loading them into a high-pressure freezer (HPF), and then operating the device. If equipment for high-pressure freezing is not available, fission yeast can be frozen by plunging a monolayer of cells into a liquid cryogen, usually ethane or propane. Unlike the HPF, where relatively large volumes of cells can be frozen in a single run, plunge freezing requires cells to be dispersed in a layer <20 µm thick. Unless frozen cells are to be imaged in the vitreous state, they must be fixed, dehydrated, and embedded for subsequent study by transmission electron microscopy; warming frozen cells without fixation badly damages cell structure. Fixation is best accomplished by freeze-substitution, a process in which frozen water is removed from samples by a water-miscible solvent that is liquid at a temperature low enough to prevent the cellular water from recrystallizing. Low concentrations of chemical fixatives and stains are generally added to this solvent such that they permeate the cells as the water is replaced. The activity of these additives is quite limited at the low temperatures required for minimizing ice crystal formation, but they are in the right place to react effectively as the cells warm up. Step-by-step protocols for HPF, plunge freezing, and freeze-substitution are provided here.

Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue.

Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in ∼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.

Improved Technology for Focused-Ion-Beam Thinning of Tissue for Cryo-Electron Tomography. Application to Triad Junctions

The use of focused-ion-beam milling (FIB) to thin vitreously frozen cells for application of cryo-electron tomography is an emerging technology. However, successful application of cryo-FIB milling and tomography to frozen biological tissue has, to our knowledge, not been reported. This is because bulk tissue cannot be effectively frozen by simple plunge freezing of EM grids, but rather must be prepared by high-pressure freezing, which requires extra steps. The cryo-transfers that are required at each step of the process can cause devitrification, excessive frost contamination and mechanical damage, consequently resulting in unacceptably high failure rates. To address these problems, we have devised a protocol and have designed new devices for manipulating the specimens as they are transferred from the high-pressure freezer, to a cryo-ultramicrotome (for rough trimming), to the FIB instrument, and finally to the transmission electron microscope. Using these improved procedures we have thinned toad fish swim bladder muscle to less than 150 nm, which is necessary to achieve resolutions of 2-3 nm, and we have determined the first cryo-tomograms of vitreously frozen triad junctions. The tomograms should reveal the three-dimensional architecture of the triad with an unprecedented fidelity and level of detail. (Supported by NIH).

High-Resolution Imaging of Dried and Living Single Bacterial Cell Surfaces: Artifact or Not?

When studying highly resolved scanning electron microscope images of cell surfaces, the question arises, whether the observed patterns are real or just artifacts of the cell preparation process. The following steps are usually necessary for preparation: fixation, drying, and metal coating. Each step might introduce different artifacts. Clever techniques have been developed to dry cells as gently as possible, for example critical point drying with different organic solvents and CO2. Instrument manufacturers also have taken account of this issue, for example, through the realization of the environmental scanning electron microscope (ESEM), operating with a low-vacuum environment saturated with water so that samples might stay hydrated. Another approach is the extreme high-resolution scanning electron microscope (XHR SEM), where the electron beam is decelerated shortly before reaching the sample. This technique requires no metal coating of the sample. Cryo-SEM also may be used, where no sample preparation is required beyond freezing in a high-pressure freezer or other cryo-fixation device. Then the cell can be examined in the frozen, hydrated state using a cryostage. However, at least some kind of preparation is necessary for SEM imaging, and we wanted to find out what changes the preparation makes on the cell surface.

A review of high‐pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues

In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4-5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.

Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high‐pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high-pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high-pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra-structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.

The Spatial Distribution of Water in Soap

Soap bars can be viewed as pseudo-binary two-phase composites consisting of solid particulate suspended in water-enriched medium. Understanding the size and the distribution of the two phases in a bar is of interest to cosmetic industry. Electron Energy Loss Spectroscopy (EELS) combined with Scanning Transmission Electron Microscope (STEM) has been used to perform water mapping in certain biological systems. Here we report preliminary analysis of soap-water matrix in a soap bar by employing EELS and spectrum imaging techniques.Thin slices of soap bars containing 13% water (as measured using Karl Fischer titrator) were frozen using a Leica high-pressure freezer (EM PACT). 200 nm thick cryo-sections were cut at -160°C. The frozen sections were cryo-transferred at -170°C to a Philips CM20 FEG electron microscope equipped with the Gatan CCD spectrometer ENFINA interfaced to an Emispec Vision system for digital control and data acquisition.

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.

A study of fat and air structures in ice cream

Three ice cream mixes of conventional composition with varying emulsifier content (no emulsifier; 0.15% mono- and di-glycerides; 0.15% mono- and di-glycerides plus 0.06% polysorbate 80) were frozen using three different freezing regimes (continuous freezer at low and high back pressure and batch freezer) in order to prepare a series of ice cream samples with varying levels of fat destabilization and foam structures. The hardened samples were viewed by thin-section transmission electron microscopy after freeze-substitution and low-temperature embedding. The images were compared to those obtained by low-temperature scanning electron microscopy. The structures created by increasing levels of fat destabilization were observed as an increasing concentration of discrete fat globules at the air interface and increasing coalescence and clustering of fat globules both at the air interface and within the serum phase. However, air interfaces at the highest levels of fat destabilization were not completely covered by fat globules, nor was there evidence of a surface layer of free fat. Air interfaces from continuous and batch freezing were similar.

High-Pressure Frozen Mouse Lung: Cryo Scanning Electron Microscopy

Abstract High-pressure facilitates freezing of biological specimens: pressurizing to 2000 bar before cooling retards growth and reduces size of ice crystals in the specimen. Although the lung is 80% air and is generally difficult to freeze, we report here good cellular ultrastructure in high-pressure frozen lung when: 1) the lung is allowed to collapse (empty of air) prior to sampling, or 2) air-filled lung is allowed to compress during pressurization in the high-pressure freezer prior to freezing or 3) airspaces are filled with relatively incompressible fluorocarbon liquid. Small Balb mice (28-30g) were anaesthetized and ventilated via endotracheal tube. The chest was opened and a 2mm diameter disk of lung at the thin anterior margin of the right lung was clamped between two aluminum tophats (200μm wells), sealing in air. In other experiments, the lung was allowed to collapse to 0 cm water transpulmonary pressure prior to clamping. In the third series of experiments, air was gradually replaced with fluorocarbon liquid (FC5312, 3M, Minneapolis MN) and the lung inflated with liquid to functional residual capacity. The top hat assembly containing the sealed lung was transferred to the high-pressure freezer (HPM 010, Bal-Tec, Balzers, Lichtenstein) where they were pressurized and frozen.

Micro- and Cryo-Techniques Prevent the Loss of Structural Information. EM-Studies on High-Pressure Frozen Tissues, Suspensions and Cell Monolayer.

Cells are information driven systems. Cellular information is stored in certain molecules, at certain places, in a certain concentration, at a particular time and under given physiological conditions. The goal of biological electron microscopy is to provide this information network, to correlate the cellular ultrastructure and its function. In this sense, it is essential to combine the high resolution of our electron optical instruments with a high information density of the biological system. Most of the structural information is lost in the course of the different preparation steps prior to electron microscopy. For this reason it is necessary that all preparation steps such as: 1. sampling: e.g. excision of tissues, 2. cryoimmobilisation, 3. follow-up procedures: e.g. freeze-fracturing, freeze-substitution and embedding, should have identical high quality levels preventing or minimizing the loss of structural information. To this aim our methodological activities focus on the development of special micro-techniques for the sampling of: a) native tissues, with an automatic fine-needle biopsy technique (1), of b) suspensions, with a special cellulose capillary technique (2), of c) cell monolayer, with a thin film cultivation technique (3) and the application/perfection of cryotechniques (high-pressure freezing (HPF) and freeze-substitution). In particular, the high-pressure freezer (HPM 010, Bal-Tec) has proven to be a highly useful tool for successful cryoimmobilization of almost any kinds of cells and tissues, bulk specimens (&lt; 200 μm in thickness) being included. This freezing technique does not require any cryoprotection, and if combined with our micro-techniques the risk of inducing artefacts as a result of specimen preparation prior to freezing is minimized.

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution.

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog (Rana pipiens) retina was sliced at a thickness of 150 or 350 microns, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO4 and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-micron slices or 15% low-MW dextran for 350-micron slices) were required for adequate freezing. The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-micron slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers. This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.

Jet freezing of cells and tissues with and without cryoprotectants

For the past several years cryofixation/freeze-substitution techniques have become valuable alternatives to chemical fixation of biological specimens. The superiority of cryofixation in preserving labile cell structures has been documented in several studies. Commercially available jet freezers and the BAL-TEC HPM010 high pressure freezer have extended high quality cryofixation from monolayer cells to cells relatively deep inside tissues. High pressure freezing can theoretically freeze biological materials of 0.5 mm thickness without the use of cryoprotectants and propane jet freezing is reported to freeze biological samples up to 40 μm in thickness without cryoprotection. Although high pressure freezing is the obvious method of choice for freezing large biological samples, its high cost combined with its apparent inability to consistently preserve microfilaments in some plant cells has prompted us to explore the capability of jet freezing to yield well frozen samples with and without cryoprotectants.We used the commercially available jet freezer JFD 030 (BAL-TEC) to obtain our results. Tightly pelleted cells sandwiched between 0.1 mm thick copper specimen carriers normally froze well without any cryoprotectants, after propane jet freezing (Figs. 1-2).

High-Resolution stereo imaging of frozen-hydrated biological samples by Cryo-SEM

The introduction of cryo-techniques to SEM allowed the biologist to examine frozen-hydrated specimens at low temperatures on a cryo-stage in the SEM. The problems associated with conventional cryo-SEM included ice crystal formation caused by low cooling rate, poor resolution, limited magnification (&lt;10 kX), specimen contamination, beam damage and charging. Recently, the effectiveness of this technique has been improved because a number of new cryo-preparation instruments have become commercially available. The high pressure freezer permits cryo-fixation of large biological specimens (diameter up to about 1mm) without ice crystal artifacts. Cryo-coating units permit fine-grain metal coating of fractured specimens. In addition, microscopes combining a short focal length lens with a fieldemission (FE) source permit high resolution imaging at low beam voltage (Vo). However, for high resolution cryo-SEM, especially for producing stereo pictures, the artifacts caused by electron irradiation remain a critical problem.The purpose of this work was to evaluate the extent to which beam damage varies with Vo, and to determine the most appropriate Vo for stereo imaging.