Freezing and drying of biological tissues with a toggle-link helium freezer and an improved freeze-drying apparatus: Application to neuropeptide immunocytochemistry

A freezing apparatus has been developed for bringing blocks of tissue into contact with a block of sapphire chilled to 17°K. A toggle linkage minimizes rebound by slowing the rate of approach of the tissue to the cold surface to a velocity of zero. A glove box limits condensation on the surface of the sapphire, and a miniature moist chamber protects the specimen from drying and premature freezing. About 50 blocks of tissue can be frozen in an hour and a half by using 5 liters of liquid helium. The tissue is then frozendried at controlled temperature, fixed with OsO₄ vapor, and infiltrated with epoxy resin in a simple bench-top freeze-drier without breaking vacuum. About two-thirds of the blocks are useful for electron microscopy. Brain tissue frozen and dried by using these methods retains enough immunoreactivity for enkephalin in plastic sections to permit its detection with immunohistochemistry by using both the light microscope (with immunofluorescence) and the electron microscope (with colloidal gold).     

Improvement to critical point drying technique for SEM

We have developed an improved method for dehydration and critical point drying which leads to a marked reduction in morphological artefact in at least two classes of problematical specimen: Foetal enamel and avian embryonic heads. Water is replaced by ethanol and ethanol by C₂C1₃F₃ by refluxing in a Soxhlet apparatus. Containers are designed to prevent air drying on transfer to the CPD bomb. We concluded that the thorough removal of both water and ethanol prior to CPD can reduce the types of artefact associated with post-CPD shrinkage (superdrying).     

Quantitation of shrinkage during preparation for scanning electron microscopy: Human lung

Human lung tissue is found to shrink considerably with preparation for SEM. Fifty-one blocks of glutar-aldehyde-fixed and inflated lung, approximately 2.5 cm × 2.5 cm × 1 cm, shrank a mean of 19% (± 4.0% SD) linear dimension through post fixation, dehydration and critical point drying. Shrinkage with fixation was not measured. Blocks of lung were observed to shrink equally in length (L) and width (W), L = 19.4% ± 2.7 SD, W = 19.0% ± 4.0 SD. Final shrinkage was the same whether samples were dehydrated in acetone or ethanol, although with acetone more of the shrinkage occurred during the dehydration process and less occurred during critical point drying.     

Ultrastructural preservation of nuclei and chromatin: improvement with low-temperature methods

The ultrastructure of chromatin has been examined in nuclei prepared by a variety of low-temperature methods. Embedding glutaraldehyde (GA)-fixed nuclei in Lowicryl K4M or K11M following dehydration by the progressive lowering of temperature (PLT) method, or in K11M following spray freezing and freeze substitution (FS), produces chromatin fibres that have, in situ, a diameter close to the in vivo state, and show internal structural details consistent with patterns of nucleosome packing previously observed only in preparations of isolated fibres. This is a temperature-dependent effect; fibres conventionally dehydrated and embedded in Lowicryl at 0°C or in conventional epoxy resin at 60°C have lower and less uniform diameters, and lack internal structural details. Of the techniques used, spray freezing followed by FS resulted in the most notable improvement over conventional methods. Inclusion of GA during FS of rapidly frozen, unfixed nuclei in methanol does not result in cross-linking of nuclear proteins. In acetone, however, cross-linking by GA occurs at — 45°C, or at lower temperatures if the water content of the acetone-based FS media is kept deliberately high. Substitution regimes employing GA alone or in combination with uranyl acetate and/or osmium tetroxide do not result in fibre morphologies comparable to either prefixed or unfixed nuclei substituted in additive-free substitution media. Whole fibroblasts show excellent preservation of nuclei and the nuclear/cytoplasmic interface after spray freezing followed by FS and low-temperature embedding.     

An inexpensive device for freeze drying and plastic embedding tissues at low temperatures

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl® and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.     

A freeze-fracture replication apparatus for biological specimens.

A freeze-fracture apparatus of original design has been constructed which can be fitted onto a standard vacuum evaporator unit. In it, cell suspensions and organized tissue may be processed by inserting a sample into a cylindrical holder. By leaving a small part of the tissue protruding from the holder, pre-selected and aligned portions of the specimen can subsequently be revealed by fracture under vacuum. After rapid freezing, the specimen remains firmly attached to the inner wall of the sample holder, preventing its possible loss during fracturing. A mechanism, in the form of a double-sided converging wedge, which is operated from outside the vacuum chamber, is used to produce a fracture in the specimen. The device gently induces a fracture in the desired part of the tissue and lifts the protruding part of the specimen out of the way. In this way, reasonably flat fracture faces are produced for subsequent replication. As the fracturing mechanism comes into contact only with the outer edges of the specimen, damage and contamination liable to occur when the entire specimen is traversed by a blade, is avoided. In addition the specimen stage is surrounded by a cold metal shroud which acts as an efficient trap for contaminants. In this way, favourable vacuum conditions are produced in the vicinity of the specimen. Such effective enclosing of the specimen also facilitates controlled sublimation of the sample.     

Preparation of Biological Material For X-Ray Microanalysis of Diffusible Elements

The appearance of ultrathin, dry-cut cryosections of brown adipose tissue and liver was found to be strongly dependent upon adequate freeze-drying. If freeze-drying was inadequate, diffusion of substances could be demonstrated and freezing damage was not apparent. Diffusion was manifested as an electron dense film over structural features such as triglyceride droplet profiles and the edges of the section; when this film was thick, X-ray signals for P, S, Cl, K and Ca could be detected from it, in different proportions to those found in the section. The frequency and intensity of diffusion were lowered by decreasing the temperature in the cryochamber from about 200 K ± 5 K to 163 K ±5 K by forced evaporation of liquid N₂ using an extra heater. The lowest incidence of diffusion was obtained in conjunction with this device, by leaving the sections in the cryochamber for at least 2 h during drying, either over a drying agent or under moderate vacuum. Such sections showed a narrow zone (A) at the edge that lacked obvious ice-crystal damage, a thicker zone (B) of moderate ice-crystal damage and the bulk of the interior (zone C) severely damaged by freezing. Qualitatively different, reproducible X-ray spectra could be obtained from ultrastructural features even in areas showing some signs of diffusion.     

A gas sensor made from a graphite paper with a nanotube film

A technique for obtaining gas sensor samples from graphite paper with a nanotube film is described. Current-induced annealing of the graphite paper and additional evaporation of a nanotube graphite film in a hydrogen atmosphere are combined in the developed technique. The current?voltage characteristics of the samples have been measured at room temperature in air, in vacuum, and at low concentrations of NH₃, ethanol, and acetone. Experiments demonstrate that these samples containing carbon multiwall nanotubes can be used as a gas sensor to detect the presence of NH₃ and acetone. They are characterized by high sensitivity and selectivity, fast response, restoration, and stability of the characteristics. The estimated sensor sensitivities to NH₃, acetone, and ethanol at a current of 96.8 mA are ~15, ~12, and ~1 mV/Torr, respectively. Their sensitivity is determined by the difference in the behavior of their current?voltage characteristics under exposure to NH₃, ethanol, and acetone. The sensor features fast response (5–20 s) and restoration (within 5 min, restoration to the initial state before the exposure to NH₃ is 100.2%), as well as the stability of its characteristics (the pressure ranges from 1 × 10^–6 to 760 Torr).     

High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Phase separation in alcohol/liquid carbon dioxide solvent systems facilitates critical point drying

Carbon dioxide and methanol or ethanol, although miscible, form alcohol/CO₂ solutions that do not easily mix with additional pure liquid CO₂. If the CO₂ inlet is situated at the top of a critical point drying apparatus chamber, pure CO₂ will entirely displace the alcohol/CO₂ phase (which is more dense) while keeping the chamber filled with liquid. This unexpected phenomenon is invaluable in critical point drying delicate biological tissues which remain continuously immersed, avoiding surface or convection currents. By providing an objective criterion for intermediate solvent displacement, the protocol also eliminates ambiguous ‘flushing’ steps.     

High-pressure freezing for immunocytochemistry

Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross-linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample-stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well-frozen material obtainable to ≈ 15 μm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystal-artefact-free freezing of samples up to 200 μm in thickness. There have been few reports of high-pressure freezing for immunocytochemical studies and there is no consensus on the choice of post-freezing sample preparation. A range of freeze-substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin-embedding, polymerization and immunocytochemical labelling. Freeze-substitution in acetone containing 2% osmium tetroxide followed by epoxy-resin embedding at room temperature gave optimum morphology. Freeze-substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl-embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze-substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice-crystal damage in sections of embedded material. Thus a slow warm-up to freeze-substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze-substitution media from immunocytochemical labelling experiments.     

An improved cryo-jet freezing method

A new cryo-jet freezing apparatus is described that is easy to use and gives good results using a propane-butene mixture (3: 1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.     

Rapid freezing,cryosectioning, and x-ray microanalysis on cardiac muscle preparations in defined functional states

Electrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosections are cut with a LKB cryoultramicrotome at temperatures of −130 to −140°C, freeze-dried at 10⁻⁶ Torr vacuum and carbon-coated before analysis. The freeze-dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMol/kg d.w.) of Na, Mg, P, S, Cl, K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x-ray microanalysis can be successfully applied to heart physiology.     

Cryofixation and ultra-low-temperature freeze-drying as a preparative technique for TEM

We have developed cryofixation and ultra-low-temperature molecular distillation drying as a method for preparing biological samples for electron microscopic analysis. To validate this approach, we have investigated the relationship between the drying characteristics and ice phases present within frozen samples. Two sample types were investigated. In the first, pure deuterium oxide (D₂O), or heavy water, was vapour condensed under vacuum conditions onto a gold-coated copper sample holder held at ?175 or ?110°C. Additionally, D₂O was slow-rate cooled from room temperature under an ultra-pure dry nitrogen gas atmosphere. The second sample type was rat liver biopsies from animals after 5 days of feeding with D₂O loaded water and ultra-rapid cooling by metal-mirror cryofixation. Ice forms present in the latter samples, determined by electron diffraction of frozen-hydrated cryosections, were amorphous, cubic, and hexagonal. Drying of samples was achieved using a molecular distillation configuration with continuous, microprocessor-controlled sample heating. The vacuum contents of the drying column were monitored by residual gas analysis (RGA) throughout the drying cycle. D₂O vapour in the vacuum chamber, as analysed by RGA, was found to increase in a phasic manner across a broad temperature range. These phases had characteristic onset temperatures and could be removed sequentially. For condensed D₂O samples, these onset temperatures were — 160, — 148, — 125 and — 90°C. Rat liver samples also demonstrated phasic drying patterns which were more complex than those detected with pure D₂O samples. Ultrastructural analysis of samples cryofixed and dried in this manner demonstrated a morphology consistent with the ice phases demonstrated in the frozen-hydrated cryosections. This, together with the RGA results, suggests the absence of devitrification or ice crystal growth during the drying procedure.     

Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy

This paper describes the results of experiments in which the volume changes in mouse embryo limb samples were followed more or less continuously after fixation through dehydration and critical point drying, with in some instances data relating to post critical point drying shrinkage. 14 and 15 day p. c. mouse embryos were fixed in 3 % glutaraldehyde in cacodylate buffer and stored in this fixative until use. Single specimens were studied using a Quantimet image analysing computer to record the changes in projected area of the unmounted specimens as they were passed through the usual series of reagents according to various commonly used dehydration schedules. The area changes were converted to volume changes for the purposes of presentation in this paper. The Quantimet system could not be used to follow volume changes in the CPD bomb so that most experiments detail the volume in the intermediate fluid before CPD and the size of the specimen immediately after it was removed from the CPD bomb. A few experiments were conducted in which the specimens were measured whilst they were in the CPD bomb. The measurements relating to dehydration and CPD procedures were compared with measurements of air dried and freeze dried specimens. All three drying methods cause considerable shrinkage: freeze drying to 85 % of the glutaraldehyde fixed tissue volume; critical point drying to 41% (after 24 h); and air drying from a volatile solvent to about 18% of the fixed tissue volume. Air drying from water caused a shrinkage to about 12% of the original volume. There was no significant difference between the various commonly used CPD schedules or between GA only and GA + Os O₄ fixed tissue. CPD via cellosolve and CO₂ caused substantially more shrinkage than other methods. Dimensional changes during specimen preparation are probably associated with changes in shape and in relative relationships between organelles, cells and tissues having different compositions. This should be borne in mind by all those interpreting scanning electron micrographs of dried animal soft tissue specimens.     

Lipid losses during processing of cardiac muscle for electron microscopy

The lipid retaining properties of several methods of processing tissue for electron microscopy (EM) have been assessed quantitatively. Guinea-pig hearts were perfused in vitro at 37°C with ³H-oleic acid bound to albumin. The hearts were fixed by perfusion with 4% glutaraldehyde in 0.1 M cacodylate buffer. Pieces of left ventricle and interventricular septum were removed., weighed and processed for EM. The fluids used at each stage of processing were monitored for loss of radioactive lipid by scintillation counting. Lipids were extracted from the processed tissue immediately before the embedding stage using a mixture of chloroform: methanol (2:1 v/v). Counts from processed tissue were compared with counts from tissue extracted directly after perfusion fixation in order to monitor subsequent losses during processing. A modified version of Epon processing, omitting 100% ethanol, acetone or propylene oxide, gave a lipid retention of only 20.6%. The addition of para-phenylenediamine to the procedure did not improve the retention although this has been shown to be a useful stain for intracellular lipid. Water soluble Durcupan which does not involve ethanol or acetone dehydration has an average retention of 63% with 100% recovery while the lesser known polymer GACH, a mixture of glutaraldehyde and carbohydrazide used both for dehydration and embedding showed a lipid retention of 82% of the counts recovered although recovery was only 69%. An attempt was made to determine which classes of lipids were present in the tissue after perfusion fixation using thin layer chromatography. It was found that the presence of any of the processing fluids affected the polarity of the lipids and their rates of migration on thin layer plates.     

Liquid nitrogen-based quick freezing: Experiences with bounce-free delivery of cholinergic nerve terminals to a metal surface

The limitations of chemical fixation in permitting the 1:1 quantitative correlations required for convincing ultrastructural explanations of cell biological processes are noted. We describe techniques for obtaining highly reproducible direct quick freezing on the polished surface of pure copper bars dipping into a static dewar of liquid N₂. The importance and the ease of testing and obtaining bounce suppression with commerically available equipment is emphasized. Artefacts caused by tissue damage and bad freezing are illustrated, and a hitherto unrecognized population of presynaptic membrane attached vesicles is described in Torpedine electric organ. Between 15 and 20% of the synaptic vesicles are attached to ca. 30% of the cytoplasmic face of the presynaptic terminal membrane. There is a close correlation between the occurrence of such attachments and the application of electrocyte basal lamina to the external face. We suggest that these vesicles are the ‘membrane operators,’ ‘vesigates,’ and ‘highly active subpopulation’ of vesicles whose existence has been invoked to explain biochemical data in other laboratories. We further speculate that relatively selective Ca pumping by this immediately submembranous population leads to displacement of acetylcholine (ACh) and reloading with newly synthesized ACh. The preferential release of the latter would then be expected.     

Preparation of animal tissues for surface-scanning electron microscopy

This paper surveys common problems associated with the preparation of animal tissues so that natural or artificial surfaces may be studied in the scanning electron microscope (SEM). Problems arise because (1) we need to prepare surfaces free from extraneous solids in solution (e.g. mucus, blood, tissue fluid); (2) surfaces of simple sections generally do not reveal significant information so that other methods of ‘opening up’ internal organization must be developed; (3) we need to remove the preponderant tissue component—water—without significantly altering the remaining structure, which generally has a very low density, is fragile and is non-conducting. In the case of natural, free surfaces to soft tissues, isolated cells or cultures the most useful approach seems to be (a) to wash with a suitable isotonic medium; (b) to fix with an aldehyde; (c) to harden with Parducz's (1967) OsO₄, HgCl₂ fixative. Artificial surfaces revealing details of internal organization are best prepared by dissecting and ripping aldehyde-perfused and fixed tissue; the duration of fixation has been found to influence the location of the ripping planes in CNS. Enzymatic digestion may usefully be combined with dissection, but must be conducted before fixation. Wet tissue surface is least distorted following freeze drying from a non-polar solvent (e.g. amyl acetate) which has substituted the tissue water. Critical-point drying requires simple apparatus and is also generally satisfactory, but it does cause some bulk shrinkage. Specimens are best coated with a combination of carbon and gold to render them conducting: carbon because it is easily scattered and thus tends to cover very rough surfaces, and because it is tough and stabilizes the surface; gold because it is the most convenient heavy metal having high secondary-electron emission to evaporate. Dried soft tissues have very low bulk densities, and beam-penetration artifacts (bulk charging and edge artifacts) are therefore troublesome. These may be limited by working at as low an accelerating beam voltage as can be tolerated to achieve the required resolution. The preparation and examination of stereo-pair micrographs is recommended as a routine because images can then be interpreted in terms of topography alone, and brightness variations of artifactual or unknown origin can be mentally excluded.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.     

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 μm, 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-μm slices or 15% low-MW dextran for 350-μm slices) were required for adequate freezing.
The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-μm 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.