On estimating freezing times during tissue rapid freezing

For the study of morphological changes that are associated with fast physiological processes, it is important to know the times at which the surface regions of specimens are frozen during rapid freezing. A simple physical model has been used to estimate the freezing times and the cooling rates at 10 μm depths in specimens. The calculations indicate that cooling rates in excess of 4 × 10⁴ K s^?1 are associated with freezing times of less than 0.5 ms. Using the same model, experimental measurements of freezing times at much larger depths have been extrapolated to a depth of 10 μm, the times obtained are 0.1-0.6 ms for freezing by rapid immersion in cryogenic liquids, and 0.1 ms or less for freezing on a metal block. It is concluded that the delay time between contact with a cryogenic source and specimen freezing is less than 0.5 ms. The uncertainty in the time of freezing may be larger than this, because of an uncertainty of about ± 0.5 ms in determining the exact time of contact and, for freeze fracture studies, because of an uncertainty of up to 0.5 ms due to imprecision in the depth of fracture. At the same time it is estimated that the time during which freezing takes place may be as high as 250 μs, which can be taken as an upper limit for the resolution time for rapid freezing.     

Rapid freezing and electron microscopy for the arrest of physiological processes

An apparatus for the rapid freezing of tissue is described, which can be used for the electron microscopy of arrested physiological processes. The material is frozen by bringing it in contact with a silver surface cooled to liquid nitrogen temperature at reduced pressure. The freezing surface is protected from condensation of moisture and gases from the air by a flow of helium gas. The cooling of the specimen during its descent through the cold helium is not large enough to interfere with physiological processes. Freezing occurs very rapidly in the surface but is retarded to about 8 msec at a depth of 10 μm. The apparatus was used to freeze frog muscle during contraction.     

Comparison of conventional and microwave-assisted processing of mouse retinas for transmission electron microscopy

Conventional fixation and processing of mammalian retinal tissues for transmission electron microscopic (TEM) examination is slow and produces ultrastructural artefacts in the photoreceptor cell layer. Among these artefacts are gaps between photoreceptor outer segment disc membranes and between photoreceptor cells in the region of the retina where the cell nuclei are located. A study was undertaken to determine whether a much more rapid microwave‐assisted fixation and processing protocol would have an effect on the quality of ultrastructural preservation of the retina, particularly on the photoreceptor cell artefacts. The overall ultrastructural preservation of the retina was similar for the conventional and microwave‐assisted techniques. However, the magnitudes of the photoreceptor artefacts were significantly reduced when microwave irradiation was used during primary fixation and processing. It is clear that, at least for the retina, employing microwave irradiation during specimen preparation for TEM results in superior ultrastructural preservation with a substantial reduction in the time required for sample preparation.     

Cryofixation of plant tissues without pretreatment

Root tips from Sorghum and Dahlia were frozen without cryoprotection by dipping into nitrogen slush, rapid immersion in liquid propane and by the high-pressure method. Structural preservation of the samples was studied using freeze-fracture (FF) and freeze-substitution (FS) techniques for electron microscopy. It was found that most of the organelles were disrupted by freezing in nitrogen slush and that only the boundary beween the cytoplasm and the vacuole remained visible. If the samples were frozen by rapid immersion in liquid propane, small membraneous organelles, such as dictyosomes, were preserved in peripheral regions of the rhizodermal cells up to 10 μm below the surface of the tissue. Specimens frozen by the high-pressure freezing technique showed good ultrastructural preservation throughout the tissues up to a depth of more than 100 μm.     

Optimum conditions for cryoquenching of small tissue blocks in liquid coolants

Three approaches were taken with the aim of defining the optimum conditions for rapid cryopreservation in liquid quenchants. In a theoretical approach, two mathematical models were used. The first is of value in defining the absolute maximum rates of cooling which could be achieved at various depths in the tissues. The second highlights the poor thermal properties of liquid coolants and therefore emphasizes the essential requirement for vigorous quenchant mixing and rapid specimen entry. Experimental work with thermocouples showed that fastest cooling rates occur at the leading edge of the object entering coolant. Of five liquid quenchants investigated, cooling rates were in the order, propane> Freon 22> Freon 12> liquid nitrogen slush> liquid nitrogen. Other considerations, however, may affect the choice of quenchant. For a given quenchant, cooling rate is maximal near the equilibrium freezing point. The consequences of quenching in the presence of thermal gradients either within the coolant or in the gas layer above it are shown. Cooling rate was found to be approximately proportional to entry velocity at least up to ～2 m s^?1 in our system. Stereological analysis of rapidly quenched, freeze-substituted tissue samples, of geometry which imposed an approximately unidirectional heat flow, revealed four zones: (i) a narrow surface layer (～10 μm) of low image contrast and apparent absence of ice crystals; (ii) a zone of enhanced contrast with ice crystals whose size increased rapidly with depth from the surface (the ‘slope’); (iii) a sharply defined zone (the ‘ridge’) of maximum ice crystal size beyond which there is (iv) an extensive ‘plateau’ with smaller ice crystals and no marked increase in size with depth. The ‘ridge’ of maximal ice-crystal damage was consistently found but varied considerably in depth from the surface (～25–120 μm) between samples. The existence of the deeper plateau region of relatively uniform ice-crystal-size may be of significance in X-ray microanalytical studies of physiological processes at some depth from the sample surface. In terms of our present understanding of the quenching process, the conditions for optimal cryofixation of small tissue samples are listed.     

Validation of convection-limited cooling of samples for freeze-fracture electron microscopy

Rapid freezing is the most important step in sample preparation for freeze-fracture and other cryotechniques for electron microscopy. A simple heat transfer model is experimentally validated to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples [Biot modulus, (hd/k) < 1] by measuring cooling rates in a variety of samples, materials, and cryogens. In comparison to the commonly accepted conduction-limited model, the convection-limited model predicts, and our experiments show, that cooling rates are proportional to the surface area to volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. We show that almost any material can be frozen at similar rates if the sample thickness, the cryogen, and the method and velocity of contact with cryogen are similar. Liquid ethane or propane cooled to liquid nitrogen temperature are shown to give the best results.     

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).     

Quick-freeze,deep-etch replication of cells in monolayers

We have made several technical improvements for quick-freeze, deep-etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly-L-lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick-freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (< 10 μm). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table–one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.     

Preservation of EDTA-expanded grid-mounted chromosomes and nuclei for electron microscopy using a specially designed freeze-dryer

We describe methods for freezing and drying EDTA-expanded, fixed metaphase chromosomes and nuclei, attached to grids as whole-mounts, for transmission electron microscopy. These methods use a special apparatus that is simple to construct. While separate freezers and dryers are commercially available, one for freezing blocks of tissue by slamming them against a cold metal surface, and the other for vacuum drying the frozen tissue, our apparatus is designed for gentler, cryogenic liquid plunge freezing and drying, sequentially, in the same apparatus, thus avoiding any compression or damage to the sepcimen. Use of a cryoprotectant is not essential; however, good results are obtained more often when 20% ethanol is used. Freezing is accomplished by rapid propulsion of the grid, with specimens attached, into slushy N₂ (-210°C) within the drying chamber; drying is automatic, by either sublimation under vacuum or by solvent substitution using absolute ethanol followed by acetone, which, in turn, is removed with a critical-point dryer. The apparatus offers a means of drying chromosomes and nuclei in an expanded state, and avoids the shrinkage of these structures that occurs during stepwise passage through increasing concentrations of ethanol or acetone.     

Time-resolved cryotransmission electron microscopy

We describe a new technique, time-resolved cryotransmission electron microscopy (TRC-TEM), that can be used to study changes in microstructure occurring during dynamic processes such as phase transitions and chemical reactions. The sample is prepared on an electron microscope grid maintained at a fixed temperature in a controlled atmosphere. The dynamic process is induced on the grid by a change in pH, salt, or reactant concentration by rapid mixing with appropriate solutions. Alternatively, induction is by rapid change of specimen temperature, or by controlled evaporation of a volatile component. We call such procedures on-the-grid processing. The dynamic process is permitted to run for a defined time and then the thin-film specimen is thermally fixed by plunging into liquid ethane at its freezing point, producing a cryotransmission electron microscopy specimen. By repeating this procedure with varying delays between induction and sample fixation, we can observe transient microstructures. We demonstrate the use of TRC-TEM to study the intermediate structures that form during the transitions between Lα, I_II, and H_II liquid crystalline phases in phospholipid systems. We also identify several other possible applications of the technique.     

Structural changes in samples cryofixed by contact with a cold metal block

A common method of cryofixation is to bring a specimen rapidly in contact with a cold metal block. It is usually thought that during this process the surface of the specimen suffers little distortion since it freezes rapidly. Whether this is likely depends on the rate at which samples freeze compared with the speed at which the sample hits the cold block. There is some discrepancy between the published experimentally and theoretically determined freezing rates. As a contribution to this debate the distortion in cryofixed, freeze-substituted, striated muscle fibres has been investigated. In transverse sections, compression can be detected by deviations of the filament lattice from the hexagonal and used to estimate the time of freezing. Some specimens were frozen using a Gatan Cryosnapper, which freezes by catching the specimen between two nitrogen-cooled copper jaws. In addition, the speed with which the jaws close has also been determined. The results suggest that freezing of the well-preserved areas occurs in substantially less than 1 ms. This conclusion is supported by results obtained using metal-mirror apparatus in which the cushioned specimen was dropped onto a nitrogen- or helium-cooled copper block. All the specimens frozen against a cold block have a flat edge whereas muscle fibres are round. At the very edge there is evidence of structural damage as well as the more general lattice distortion.     

Natural propane cryogen for frozen-hydrated biological specimens

The properties of natural propane, mixed with 0–4% isopentane, as a cryogen suitable for rapid freezing of this layers of aqueous biological specimen suspensions are discussed. Although natural propane has rather variable properties, its freezing point can be depressed below the temperature of liquid nitrogen by adding a smaller amount of isopentane than is required for depressing the freezing point of pure propane.     

Micro-contact analysis for the initial contact in nanoindentation tests

The force-depth behavior of initial contact between a Berkovich indenter and S45C steel specimens has been examined. The indenter is considered as a rigid sphere with a radius of 300 nm since the blunt tip is dominant under initial contact. The S45C steel specimens were prepared to have different surface characteristics. The specimen surface profile was decomposed by Fourier cosine series; then the statistical evaluation for force and area of micro-contact was proposed. The influence of surface roughness on the real contact area and thus the contact pressure arising in the indentation test can be investigated from the proposed analyses. The force-depth responses obtained by the proposed method revealed good agreement with the experimental results for the prepared specimens with their different surface characteristics. The evaluated results of the force fractions in the elastic, elastoplastic, and plastic deformation regions showed that the S45C steel specimens had fully plastic deformation under the initial contact load of 5 μN. The average values of real contact pressure evaluated by the current method rapidly reached the hardness value. Through the proposed method, the dominant radii of summits were evaluated and their relation to the indentation depth was demonstrated.     

Cryo preparation of isolated rabbit pancreatic islets

A Flotronic silver membrane has been used as a vehicle to process, via freeze substitution, collagenase-derived rabbit pancreatic islets. The procedure provides: (1) a simple, inexpensive method for handling larger numbers of tightly clustered islet aggregates; (2) a metal surface for rapid heat transfer from specimen to cryogen resulting in an increased circumferential zone of fine structural preservation; (3) the elimination of possible artifacts associated with impact or rotation of biological specimens against a cooled, highly polished metal block; (4) superior preservation of structural components not usually observed by conventional modes of fixation; (5) retention of metabolic components which may subsequently be available for immunocytochemical or X-ray energy dispersive procedures.     

Freeze-drying of articular cartilage: Investigation of rat femoral heads by SEM

The results of comparative investigations of freeze-drying of joint cartilage which had not been separated from the underlying bone are reported. Fixation and ethanol and amyl acetate substitution procedures cause marked loss of ground substance and thus create surface structures which are not present to the same degree in cartilage which is simply freeze-dried. Indeed, it is questionable whether these structures are present in vivo. They were most highly developed in critical-point-dried cartilage. The main difficulty with freeze-drying is the prevention of damage caused by ice crystals. A tissue temperature of ?130°C should always be aimed for and, because of the rapid growth of ice crystals, the temperature should never be allowed to rise above ?90°C. These conditions can be fulfilled if the specimen is in good thermal contact with the cold stage and if the cooling device is equipped with a condenser which is cooled with liquid nitrogen. The better the tissue was processed, the smoother was the resulting cartilage surface and the greater was the degree to which the chondrocytes fitted the walls of their lacunae. At tissue temperatures above ?90°C, the first change which became apparent was destruction of the cell membranes. This was followed by almost complete elution of the ground substance from between the fibers and, finally, the cell nuclei were also destroyed. This damage caused by ice crystals exposed fibrous structures on the surface of the cartilage, and the appearance of these structures was superior to that produced by enzymatic methods of preparation. The disruption caused by freezing uncovered intracellular structures.     

Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans , could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.     

A quantitative cryo-scanning X-ray microanalysis protocol for the examination of the eye

Analysis of elements present in fluids contained in small, poorly accessible sections of biological tissue is challenging. The choroid of the eye, which is a vascular tissue approximately 100 microm thick, surrounds the retina for the purposes of nutrient supply and metabolite removal, and which in the chick shows dramatic volumetric change in response to visual experiences. Because fluid homeostasis is critical to good vision, a complete understanding of the ionic changes driving large shifts in ocular fluids is required. However, the structure of the choroid and retina make extraction of pure fluids for analysis extremely difficult. Elemental x-ray analysis on a transverse chorioretinal specimen was performed after rapid freezing of a whole chick eye in liquid nitrogen, and mechanically fracturing the frozen globe. Using a Polaron Cryotrans System on a Cambridge S-360 scanning electron microscope and a Kevex Quantum detector, spectra were obtained for blood vessels, lymphatic vessels and vitreous that were readily visible at 265x. Analysis was performed on a frozen control solution of the elements found in the vessels. The elements and their concentrations found in blood vessels by x-ray analysis compared well with those from whole blood as established by conventional means. The analysis for lymph yielded results compatible with expectations; no other published data for small lymphatics enable a direct comparison. In conclusion, x-ray analysis can be used to acquire information that is otherwise unobtainable from tissue in situ. The same bulk-frozen elemental microanalysis protocol would have application to other organs and tissues when access to the site would destroy the integrity of the tissue under investigation.     

Study on micro-interacting mechanism modeling in grinding process and ground surface roughness prediction

In the research on grinding process modeling, the stochastic nature of grain sizes and locations need to be considered. A new numerical model was developed which will describe the micro-interacting situations between grains and workpiece material in grinding contact zone. The model was established based on a series of reasonable assumptions, the critical conditions of starting points of plowing and cutting stages, and the redefined grinding contact zone. It indicated that there are four types of grain existing in grinding contact zone: uncontact, sliding, plowing, and cutting grains. The number of grains per unit wheel volume (N _v ) and the undeformed chip thickness (h _cu,max), which are key parameters in grinding process modeling, were firstly obtained. The numbers and distributions of different grain types along grinding contact zone were then obtained and analyzed. Calculation results showed that only a small fraction of grains participate in cutting interactions and the changing laws of each grain types along grinding contact length are very different from each other, which gives a deeper insight into grinding process and can be a good foundation for more precise grinding force prediction and thermal analysis. Another important application of this model is for ground surface roughness prediction and a new method on this purpose was developed. At last, two comparisons were made between calculation results and existing experimental data for validating the work on paper. Comparison results showed that the roughness of ground surface can be well predicted and gave the method theoretically to reduce ground surface roughness.     

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.     

Examination of mucosal surfaces by scanning electron microscopy before and after removal of debris

A rapid method is described whereby gastrointestinal biopsy specimen surfaces can be examined by scanning electron microscopy with overlying tract contents (debris) intact, and also re-examined after cleaning to determine the structure of the underlying mucosal surface. The conductive coating of gold is removed using mercury, a non-wetting agent. The specimen surface is suitably cleaned of debris after a brief ultrasonication in absolute ethanol which mixes with the transitional fluid (CO₂) used for critical point drying.