Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph.

Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1).

High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.

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 (< 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.

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.

In the interactions of Candida albicans with host cells, the cell wall of the yeast may play important roles in the adhesion of yeast cells to tissues. The outer cell wall of yeast (e.g. Saccharomyces cerevisiae, C. albicans) has been shown to consist of a dense network of radially projecting fibrils composed of mannoproteins that are known as fimbriae and which previously have required cryopreservation either by jet propane freezing or by plunge freezeing for their visualization. High pressure freezing provides an advantage over jet or plunge freezing in terms of the higher consistancey in the quality of freezing, and the minimization of formation of ice I with this method. Hohenberg et al reported a method utilizing cellulose capillary tubes to cryoimmobilize suspensions of microoganisms by high pressure freezing (HPF) and freeze substitution (FS), and herein, we describe an adaptation of this method by culturing microorganisms within the tubing to increase cell density prior to high pressure freezing and freeze substution.

In vertebrate somatic cells, sister kinetochores attach chromosomes to the mitotic spindle by capturing microtubules (Mts) emanating from opposite spindle poles. Kinetochores are also required for chromosome alignment at the spindle equator, stabilizing kinetochore Mts, cell cycle control of anaphase onset, and poleward migration of sister chromatids during anaphase. Thus kinetochores play a critical role in the distribution of genetic information to daughter cells during cell division. Understanding the molecular mechanisms behind kinetochore function requires knowing how the molecular components are arranged relative to each other, and relative to the kinetochore Mts. Currently, however, our knowledge of kinetochore composition is incomplete, and until recently our knowledge of kinetochore ultrastructure was limited to the familiar trilamellar model derived from conventionally fixed and dehydrated specimens(Figures 1a&b).

Highly fibrous structures, such as the kinetochore, are particularly vulnerable to distortions caused by the chemical fixation and dehydration methods used in conventional specimen preparations. Such distortions can be largely avoided by using high pressure freezing and freeze substitution (HPF/FS).

As the defining structure in eukaryote cells, the nuclear envelope is completely dismantled and reformed within an hour or so at each cell division (open mitosis). In yeast and some insect tissues, closed mitosis occurs, in which the nuclear envelope is maintained largely intact throughout chromosome separation. Use of cell free systems has allows us access to the mechanisms of cell division and NE dynamics in vitro by FEISEM (Field Emission In Lens Scanning Electron Microscopy. We have used demembranated Xenopus sperm heads as a source of DNA, which is incubated in an extract of Xenopus egg cytoplasm, where it becomes assembled into a normal nucleus with functional nuclear envelope (1-4). DNA replication proceeds under normal cell cycle controls, followed by an in vitro mitosis in suitable conditions. The cytoplasmic extract can be separated into membrane and soluble fractions that can be supplemented with, or depleted of, specific proteins. Inhibitors and other effectors can be .added to modulate both assembly and transport (5). Using the lectin WGA we have depleted Xenopus cytoplasmic extract of the major nucleoporins, CAN, Nup 98 and p62 and their associated proteins, whose removal effectively inhibits three aspects of nuclear formation, namely NPC formation, nuclear growth, and the reorganisation of the DNA in the depleted nuclei. Adding back these eluted nucleoporins restores normality with respect to nuclear growth, DNA reorganisation and NPC assembly. Current work involves purification of complexes containing these proteins by HPLC to allow add back of the complexes singly and in combination, to characterise their individual roles in NPC assembly, structure and transport (Figures 1,2,3).

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.

Electroporation has been used to permeabilize the skin to enhance the transdermal delivery of molecules. The mechanism is generally believed to be based on the temporary breakdown and permeabilization of the stratum corneum by the applied pulsed electric field. The kinetics of recovery are believed to be in the millisecond range. However, no structural changes within this time scale have been observed.

We studied the morphological changes to heat stripped porcine stratum corneum following an electroporating pulse by time-resolved freeze fracture electron microscopy. Pulses at a supraelectroporation threshold of 80 volts and 300 μsec were applied across the stratum corneum with a pair of copper plate electrodes, which also served as cooling contacts. The pulse timing was accomplished through the time-of-flight of a trigger bar attached to the plunging rod holding the sample.. The trigger bar passes through two optical switches. As the sample was propelled towards the liquid propane coolant container, the first switch triggered the pulse generator and the second marked the precise time at which the sample was frozen. The sample was pulsed at set times of 0.5 ms, 12ms, 5 sec, 60 sec, and 300 sec before freezing.

Cryo electron microscopy has become an important technique for studying the structure and composition of biological specimens in their frozen hydrated state. Many modern transmission electron microscopes can be modified for cryogenic analysis by incorporating liquid nitrogen (2) cooled anti-contaminator blades and a special 2 cooled cold stage specimen holder. These specimen holders are used routinely for the examination of frozen hydrated specimens, and many laboratories have several such specimen holders to permit efficient handling of multiple specimens. Typically, these devices are cooled conductively via intimate contact with a reservoir of 2 inside of a small-evacuated dewar. The periodic evacuation of these dewars is one of the primary purposes of the pumping station. In addition, a means of storing the cryo holders under low vacuum is an important design feature of the pumping station.

In our experience the commercially available unit for pumping and storing the cryo holders proved unsatisfactory in two important aspects. The commercial unit made no provision for isolating the pump vibrations from the fragile cryo holders. Occasionally, this resulted in damage to the devices and subsequent costly repairs. Economically, the mechanical arrangement of the ports and associated valves were not as convenient to operate as they could have been. The purpose of this paper is to describe a custom pumping station that was designed and built with off-the-shelf components that overcomes the shortcomings of the commercial unit.

Freeze Substitution has historically been used for processing small pieces of tissue for microscopic examination. These small pieces of tissue have usually been ultra-rapidly frozen by either metal mirror, high pressure or immersion fixation. In today's expanding field of cryobiology, freeze substitution is being employed to better understand the effects of ice formation and possible deformation of cell structure in tissues that have been processed by controlled-rate freezing. This technique is of extreme importance in laboratories focused on developing methods of preservation by freezing tissues, such as heart valves or veins, which are intended, for clinical applications.

Overview: Many laboratories use methods of substitution, which incorporate acetone as the ice dissolution solvent. We have done comparative studies using both acetone and methanol as a substitution media at -80° C and -90° C. Theoretically, either of the two methods should give satisfactory results provided one uses extremely small tissue samples and follows the accepted guidelines for substitution. Since our protocol requires the use of relatively large samples, which are encased in a voluminous amount of media it is obligatory to adhere strictly to a methanol (-90° C) substitution regimen to be assured complete substitution has taken place (Table 1). Porcine heart valves were dissected into segments consisting of the aortic wall and leaflet. Segments were exposed to a variety of new cryoprotective agents (CPAs) and placed in the center of a parafilm sleeve with 1.5ml of CPA and cooled at 1° C/min to −80°and stored in the vapor phase of liquid nitrogen. Samples were processed for either routine histological studies or freeze-substituted to obtain profiles of ice formation.

The field of cryobiology is expanding and incorporating conventional methods used in electron microscopy into protocols heretofore unexplored; two of those methods is that of ultrarapid freezing and freeze substitution. When tissues such as heart valves and/or vascular tissue is frozen for clinical applications it is imperative to know the quality of the freezing process.

Ultrarapid Freezing: Various methods of ultrarapid freezing have been employed routinely by those utilizing electron microscopy. In particular, questions involving immunocytochemistry and x-ray microanalysis may indeed require the use of one or more of these methods.. Metal mirror fixation (slamming) is the preferred method of cryopreservation for most subsequent protocols. It is also the preferred, and probably the only, method to check the accuracy of freeze substitution protocols. The metal mirror method utilizes a copper block, which is brought into contact with the tissue, at a predetermined speed, via a pneumatically operated system. The chilled metal block, devoid of scratches or imperfections, ensures maximal preservation of tissue to a depth of 15μm, which can serve as a control, for both fixation and the substitution process.

We conducted physical and mathematical modelling of the process of substituion of ice with organic solvent. The result was a relationship between the time of freeze-substitution and the sample thickness. The model is based on the solution of mass exchange problem between a porous plate (frozen tissue) filled with a solid matter (ice) and an organic solvent (alcohol, acetone) that is contained in an apparatus with an active fluid circulation. The model takes in account porousness of tissue, dissolving of ice with solvent that produces a new medium, dissolving of that medium and its substitutions with solvent after solvent concentration exceeds evtectic concentration for the given temperature. Our results (fig. 1) agree with previously published results [1].

We also conducted experiments using original device [2] that implements freeze-substitution method. The device is made of the desk apparatus, comprising cryogenic and electron parts. Cryochamber ( Fig. 2 ) contains cylindrical unit (1), central channel (2) and located around it cylindrical vessels (3), connected by means of upper (4) and lower (5) channels with the central one. Perforated containers (6) with frozen bioobjects are adjusted on metal rack (7). The flux inducer (8) provides the circulation of substituting medium. The cold-conduct or (9) is submerged into a liquid nitrogen. Temperature control is performed be the thermogauge (10). The range of operating temperatures stabilization is from +20° to −120° C.

Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context.

In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).

Recording of images under low dose conditions is a rather critical operation including many intermediate steps. Moreover, there is not a single fully agreed protocol concerning low dose operation but several competing ones potentially including individual variations. In any case, however, there should be a very well defined sequence of steps which must not allow for any mistake. Therefore tailored individual automation is the only way to obtain reliable results with maximum throughput at the same time.

Such automation requires the functional integration of a fully remote controllable digital TEM, a Slow-Scan camera with a high sensitivity, and an acquisition/remote control software providing macro capabilities dedicated to different logical cameras. These requirements are met by LEO (EF)TEMs, by the ProScan SSCCD 14 bit camera, and the analySIS software 3.0.

Three different logical cameras have to be configured in the SIS image processing software. The first camera is defined for searching for suitable specimen areas. It operates with 5 MHz and 8 bit image depth providing 1k×1k field size and resolution. The exposure time should be as short as possible, e.g. 50 ms for each frame. Via a preprocessing macro image brightness and EM magnification are set to the appropriate values, the desired shutter is activated, and the viewing screen is lifted for the exposure. A second postprocessing macro blanks the beam and does additional settings when the camera is stopped.

Colloidal Liquid Aphrons (CLAs) are composed of surfactant doped non-aqueous inner cores surrounded by thin surfactant films. These are created by intense stirring of the non-aqueous phase in an aqueous surfactant solution. Large interfacial areas, due to small size, and high stability (much greater than conventional emulsions) make aphron dispersions attractive for a variety of mass transfer limited processes. The unusual stability of CLA dispersions has been attributed to the multi-layered structure that includes an entrapped water shell around the non-aqueous core as postulated by Sebba. However, no direct evidence for the structure of the surfactant-stabilized interface surrounding the CLAs has been previously published.

CLAs were prepared as previously reported. Comparative conventional emulsions were made similarly, with the lipid phase free of the Tergitol®(15-S-3) surfactant used in the CLAs. Aliquots (≈30 μl) of the dispersions were spread on 0.15 mm thick, 10 mm diameter copper supports These supports were ultra-rapidly frozen via plunging in liquid propane(−190°C). The frozen samples were inserted onto a pre-cooled rotary stage of a freeze-etch unit (modified Balzers BA-510) and fractured by knife at 150°C with a residual pressure of approx. 6.5 x 10−5 Pa. The replicas were shadowed with ≈2nm of Pt at 30° followed by 10-15 nm of carbon by arc evaporation. The resulting replica was washed in an 10% graded isopropanol series, rehydrated, washed in chromic acid, rinsed in de-ionized H2O, and then mounted on 300 mesh grids. TEM was performed on a JEOL 100CX operated at 120keV.

The introduction of a dual Nipkow disk scanning head should facilitate confocal microscopic study of live specimens. The use of micro-lenses in this design improves the Z-axis resolution, scan speed and overall optical efficiency of the original Nipkow disk design. Advantages of this device include the capability for high speed scanning (360 scans/sec), real time viewing, superimposed transmitted light and confocal imaging, together with reliable optical alignment, and a compact modular scan head that attaches to the video port of existing microscopes. When used with low light & light amplified cameras, lower power densities can be used to illuminate live specimens resulting in longer experiments being successfully completed.

The schematic diagram shows the optical path and specifications of the new scan head. A pre-aligned collimated light beam illuminates the upper micro-lens disk array. Each lens within the array focuses light through a small pinhole aperture on the lower Nipkow disk providing confocal multi-point scanning of the specimen. Fluorescent light emitted from the specimen passes back through the pinhole apertures to the dichroic mirror located between the two rotating disks. The dichroic mirror reflects the confocal image into the detector path. The confocal image can be observed directly through a 10x ocular or relayed to the camera port. The camera is chosen to achieve the desired combination of speed, dynamic range and spatial resolution. The acquisition, display, and recording aspects of the system are matched to the camera and are readily available. Limitations of the system include a fixed Z-axis resolution determined by the objective lens and pinhole size.

The Leica TCS SP Confocal Microscope combines spectrophotometric detection with confocal microcopy. The result is a multi-channel confocal imaging spectrophotometer that significantly increases the flexibility and efficiency of the detection system.

In conventional point scanning confocal microscopes excitation light from laser(s) is delivered to the scan head via fiber optic, passed through a pinhole, reflected by a primary dichroic to the scanning system, and scanned onto the surface of the specimen. Light emitted from the specimen is descanned and passed to the detection system. The detection pinhole(s) are placed either between the primary dichroics and the detection system as in Leica confocal microscopes or closer to the end of the detection system. In the detection system emitted light is separated using a combination of dichroics, mirrors, and barrier filters before being passed to the photomultiplier detectors.

The Leica TCS SP Spectral Confocal microscope has the same beam path as the filtered system up to and including the detection pinhole. The detection system including the secondary dichroics and the barrier filters is replaced by the spectrophotometer detection system (SP). The light emitted from the focal plane that passes through the detection pinhole has both intensity and spectral information. In the SP system this light is passed through a prism. The prism splits the emitted light into a spectrum from 400 to 750 nm. The spectrum is directed at a PMT for detection. The physical dimension of this spectrum is such that the entire spectrum can be imaged onto the window of the PMT.

The use of water immersion objectives can provide a significant benefit to research involving the study of dynamic events in living tissue. Optical and quantitative imaging techniques like Confocal, Differential Interference Contrast (DIC), and Epi-fluorescence microscopy are dependent on the ability of high magnification, high numerical aperture immersion objectives to provide a high resolution image. The need to image the specimen not only at the surface next to the cover glass, but deeper down to 200 microns or more, can be greatly assisted by the use of highly corrected water immersion objectives. With oil immersion objectives, the deeper into the specimen and aqueous media one images, the more spherical aberration occurs.

Spherical aberration is the main optical problem in imaging living cells with oil immersion, and spherical aberration increases proportionately with depth into the aqueous media and cellular material. The destructive effect of spherical aberration is seen as a loss of intensity and contrast, inability to collect and resolve small spatial frequencies, and reduced accuracy of reproduction in an optical system, all in direct proportion to the distance from the cover glass. It is possible to compensate mathematically for these distortions and extend resolution with deconvolution or image restoration software. These methods require accurate measurement of the point-spread function; if resolution varies with depth, then measurements must be taken at different depths, which complicates the process.

The laser scanning confocal microscope (LSCM) has enormous potential in many fields of biology. Quality assurance is essential for the equipment to function correctly and realize its fullest potential. Currently the assessment of a confocal microscope’s performance is subjective primarily by evaluating the system with a specific test slide provided by the user. To achieve better performance, it is necessary to run a series of validation tests. These tests that were applied to measure field illumination, lens, clarity, dichroic functioning, objective spectral alignment, axial resolution, laser power and stability, photomultiplier tube (PMT) functioning, and system noise. Although th?se tests were derived on a Leica TCS-SP system and Leica TCD4D system, they should be applicable to other manufacturers’ systems.

For optimal images, it is important to control the variables of laser power, PMT tube voltage, averaging, and bleaching. The relationship between PMT voltage, laser power, and averaging was determined using a Spherotech Rainbow 10 μm diameter test bead. It was demonstrated that the noise (coefficient of variation of bead intensity, CV) in an image greatly increases as the PMT increases. The system variables that affected the precision and accuracy of the confocal system will be discussed.

We are developing a “new-generation” fluorescence microscope that will allow very fast (milliseconds) acquisition of fully three-dimensional (3D) images for a wide spectrum of biological applications. This new system will overcome the slow image acquisition constraint of existing confocal and widefield deconvolution microscopes (the two most commonly used instruments for 3D fluorescence imaging) that has prevented them from being used for investigations of live-cell dynamics in three dimensions. Our new microscope incorporates the innovative techniques of optical wavefront coding, pioneered by W. T. Cathey and E. R. Dowski, University of Colorado. With this new system, as compared to the normal sequential plane-by-plane image acquisition requirement of confocal and widefield microscopes, we need acquire only a single CCD camera image to obtain an equivalent extended-depth-of-focus (EDF) rendering of a thick specimen, and a minimum of only two images for a 3D stereo view that has full depth.

Our microscope system uses a special-purpose optical element to uniformly “code” the information from all planes throughout the specimen volume onto a single CCD camera image. Specimen-independent digital processing is then used to “decode” this raw image. In effect, the coded raw image is blurred by a special type of aberration which produces an image that is nearly independent of focus. The system then uses a fast, non-iterative, digital filtering algorithm to remove this special blur so that a large volume of the specimen image appears sharply focused all at once.