A simple and inexpensive liquid helium cooled ‘slam freezing’ device

A liquid helium cooled copper block device has been designed for the rapid freezing of biological material. The apparatus differs from previous designs in being easily constructed from readily available components. It incorporates a novel and simplified specimen carrying system. Construction of this apparatus is possible for any laboratory and makes the advantages of freeze substitution reliably available for many cell types at minimal cost.     

Frozen-surface replicas of rat bladder luminal membrane

A method for preparing replicas of the luminal surface of frozen, unfractured but deep-etched whole bladder tissue using a Bullivant type II device is described. A small piece of glutaraldehyde-fixed (uncryoprotected) rat bladder is rinsed in distilled water, mounted luminal side uppermost on a specimen holder and rapidly frozen by immersion in liquid nitrogen (cooled below its boiling point in a vacuum) or by contact with a copper block at liquid nitrogen temperature. The specimen is processed in the type II device without fracturing and 'deep-etched' by allowing a longer period than usual to elapse before shadowing. The results are assessed with reference to the appearance of the luminal membrane in standard freeze-fracture replicas, and some preliminary observations on the structure of the normal luminal membrane and its counterpart in bladder tumours are presented.     

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.     

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.     

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.     

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.     

Experiments on freezing damage with freeze substitution using moth antennae as test objects

Insect antennae can be easily frozen by immersion into propane at 90 K, freeze substituted at 194 K and, after warming up, embedded and sectioned at room temperature. Freezing damage (f.d.) may occur during cooling the specimen down (primary f.d.) or due to re-crystallization, during warming up, if substitution was not complete (secondary f.d.). Experimental evidence suggests that secondary freezing damage is not likely to occur with freeze substitution, provided the acetone is water free (by adding molecular sieve) and the rate of warming up is low. All freezing damage observed in the specimens therefore most probably is due to primary freezing damage during cooling. Propelling the specimen with high speed into the coolant, instead of merely dropping it in, does not improve the quality of preservation obtainable but increases the yield of well-frozen specimens.     

Simple procedures for evaluating the cryofixation of biological samples

Ultra-rapid cooling of biological material can be achieved in the absence of cryoprotectants by using thin samples. Three methods now employed to prepare thin samples for freeze-fracture electron microscopy are compared: contacting the sample against a liquid helium-cooled copper surface (Heuser et al., 1979), spraying the sample with a jet of propane (Mueller et al., 1980), and plunging a streamlined copper ‘sandwich’ into liquid propane (Costello, 1980). In the first method a thin surface layer of the sample is ultra-rapidly cooled while in the other methods the entire sample sandwiched between sheets of conducting metals is cooled. The morphology of fracture-faces of dilauryllecithin-water systems is used to evaluate the effectiveness of cooling methods. At optimum cooling rates the initial disordered arrangement of lipid in the lamellar (L_α) phase is preserved, giving smooth fracture faces. At slower cooling rates a worm-like texture appears which signals the formation of molecular ordering characteristic of the P_β, phase. All three methods are capable of cooling these lipid-water phases as well as other more dilute aqueous suspensions without evidence of ice crystal growth or damage. Measurement of cooling rates employing miniature thermocouples embedded in samples indicates that rates for all three methods are in excess of 10,000 K/s. The propane jet (32 times 10³ K/s, slope at 273 K) exposes the sample to coolant more rapidly than the sandwich plunging method (10 times 10³ K/s, slope at 273 K) and therefore produces slightly higher cooling rates for samples of equivalent mass and thickness. Each method has its advantages. The contact method is well suited for tissues; the sandwich method is simple and inexpensive; the jet method can potentially produce the highest cooling rates. The last two methods yield complementary replicas.     

A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension

We have developed a quick-freezing method, using a copper block cooled with liquid helium or nitrogen, which permits us to freeze muscles without any cryoprotectant at predetermined, precisely measured points in the recorded tension time-course of a single twitch or tetanus. Our aim is to arrest structural intermediates of the cross-bridge cycle for observation in the electron microscope. Chemically stimulated, demembranated muscles as well as electrically stimulated, live muscles can be frozen on the same apparatus. Good freezing of relaxed and contracting muscles has been obtained to a depth of 10–20 μm, with excellent structural preservation after freeze-substitution.     

The relative efficiency of cryogens used for plunge-cooling biological specimens

Coolants used for freezing biological specimens were tested for cooling performance in the continuous plunge mode. Results from bare thermocouples showed that ethane cooled faster than propane or a propane: pentane mixture, even when warmed to 25 K above its freezing point. Propane coolants were more efficient than Freon 22 and the slowest cooling occurred in boiling liquid nitrogen. Hydrated gelatin specimens showed similar results with ethane cooling about 33% faster than propane. Epoxy resin specimens cooled faster than hydrated gelatin specimens of similar size. Hydrated and resin specimens cooled over increasing distances as plunge velocity increased. A bare thermocouple, however, cooled over a constant distance when plunged above a critical velocity. This phenomenon may reflect vapour formation and its suppression at high plunge velocities. The rate of cooling in hydrated specimens is shown to have an absolute limit and cannot be modelled by bare thermocouples or resin specimens.     

Freeze-fracturing at low temperatures: I. A device for fracturing biological specimens at 77–10 K under high vacuum

Standard freeze-etching or freeze-cleaving is performed at 173 K in a vacuum of 133 μPa or at 77 K under liquid nitrogen with subsequent transfer of the specimen into a vacuum chamber. It has been suggested that the frequent poor resolution of morphological details, the poor complementarity of innermembrane protein particles and the semi-crystalline substructures in biomembranes are caused by structural distortion or plastic deformation due to sheer forces which occur even at 77 K during fracturing or cleaving. In addition, water contamination and radiant heat damage occurring during replication introduce artefacts to the structural record. These artefacts could be avoided or reduced by lowering the temperature at which fracturing or cleaving and shadowing is carried out, to about 10 K. Therefore, a device for cleaving biological specimens at 15–10 K under high vacuum was constructed. To allow the use of existing equipment, the device was built into a standard Balzers 301 vacuum unit, where the specimen transfer is done via an airlock system which allows hoar frost contamination free transport of the specimen holder onto the specimen table. To reduce or prevent the condensation of water and other residual gases in the vacuum onto the freshly cleaved specimen surface at 10 K, the specimen is surrounded by two cooled surfaces of 6 and 20 K. All condensable gases outside those shielding shrouds will condense on these surfaces before reaching the specimen. This makes it possible to work at a high vacuum of 3 μPa outside the cooled shrouds, which can be reached with standard turbomolecular pumps. The actual vacuum within the cooled shrouds is estimated to be approximately 13 nPa. Residual gas analysis before and during replication reveals equal conditions to ultra high vacuum systems. An analysis of the yeast cell paracrystalline plasmalemma structure shows that the topographic resolution of the crystalline arrays has been improved by working at 12 K. However, plastic deformation still occurs under these conditions. This observation points to the possibility that what is described as plastic deformation, for at least some membrane proteins, may be a loss of resilience at low temperatures.     

Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry

Cryo-fixation followed by freeze-substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze-substituted at ?80°C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well-preserved ultrastructure was observed in the outer 10–15 μm of all samples. Positive immunocytochemical localization of fixation-resistant and fixation-labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR-75-1 at both light and electron microscope levels.     

A Controlled Magnetic Clamp

A clamp for establishing a thermal contact between cryostat components in a vacuum is described. The contact is established by pressing heat conductors to each other using a permanent magnet and is broken or established by applying a current pulse of the required polarity to the winding around an iron magnetic core. The device is held in the open state by a second permanent magnet. Through the pressed contacts, a copper block with a mass of ～600 g is cooled in the vacuum from the liquid-nitrogen temperature to the liquid- helium temperature within 4–5 h.     

A new freeze-drying device for platinum replica studies of cell surface and cytoskeleton: An example using immunogold-labeled human erythrocytes

We designed and built a freeze-drying device that ensures the protection of the specimens against contaminants during mounting on the cold stage of the freeze-fracture machine, transferring into the vacuum chamber and deep etching. The device consists of a copper cap that covers the specimen and a thermal connection that ensures thermal transfer between the microtome arm and the copper cap. This device was used to study the ultrastructural features of the erythrocyte membrane skeleton and the immunocytchemical localization of spectrin in an “in situ” approach, by freeze drying and platinum rotary shadowing. Human erythrocytes adhered to polylysine-coated coverslips and were broken by a stream of buffer that mimics the intracellular ionic environment (“inside buffer”). The samples were prefixed in periodate-lysine-paraformaaldehyde fixative, labeled with antispectrin 5-nm gold particles, fixed in glutaraldehyde, mordanted in tannic acid, postfixed in OsO₄, repeatedly washed in water, rinsed quickly in 30% ethanol, freeze-dried, and rotary-shadowed. Electron microscopic examination of the replicas revealed the skeletal network on the inner surface of the erythrocyte membrane. Immunocytochemical labeling proved that spectrin represents a fibrillar component of the network. Our data confirm the speculative model of the molecular organization of the erythrocyte skeleton, based on studies on in vitro association of proteic constituents. Both the technique and the device developed by us may lead to a deeper understanding of the spatial organization of the cytoskeletal network of more complex cell types.     

A device for the rapid freezing of biological specimens under precisely controlled and reproducible conditions

The construction and preliminary testing of a device is described which can be used to freeze biological specimens in any cryogenic liquid at temperatures down to the nitrogen freezing point (63 K) and which can operate in the pressure range 1.3 kNm^?2 to 1 MNm^?2. Ultra-rapid freezing can be carried out in a subcooled cryogenic liquid either hyperbarically or at atmospheric pressure. Slow freezing rates can be achieved by cooling the specimens in a controlled manner in the vapour phase above the liquid bath.     

The direct measurement of temperature changes within freeze-fracture specimens during rapid quenching in liquid coolants

We have evaluated the cooling rates of specimens mounted in a variety of freeze-fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded. A simple guillotine-like device for quenching freeze-fracture specimens under reproducible conditions is presented.     

检测激光晶体侧面致冷均匀性的马赫-泽德干涉法

提出了一种基于马赫-泽德干涉仪的检测法。理论分析表明,该法检测到的物光与参考光的干涉条纹即为晶体径向平面上的等温线。通过热分析软件对均匀和非均匀致冷晶体温度场的模拟表明,当检测到的干涉条纹为以晶体径向平面中心为圆心的圆环时,晶体周边接触良好,致冷均匀;当检测到的干涉条纹向某一方向扭曲时,该方向上致冷块与晶体边界接触不好。对装在铜致冷座里的3 mm×3 mm×5 mm,a切割,0.5% Nd³⁺掺杂的Nd:YVO₄晶体进行了检测,根据检测到的干涉条纹扭曲方向重新安装了致冷座,获得了以端面中心为圆心的干涉环。实验结果与理论分析相符,证明马赫-泽德干涉法可以检测晶体侧面致冷均匀性。     

The cryopuncher: A pneumatic cryofixation device for X-ray microanalysis of tissue specimens

A cryopunching device is described which allows cryofixation of tissue specimens by quick contact with a precooled copper surface during excision. The advantage of the cryopuncher for analytical electron microscopy of cells and tissues in defined functional states is illustrated by electron probe X-ray microanalysis of freeze-dried cryosections from rat liver and dogfish kidney. In comparison with results obtained from specimens plunged into liquid propane, cryopunching in situ results in similar preservation of morphology and remarkably improved intracellular K/Na ratio.     

Rapid cryofixation of rabbit muscle fibres after a temperature jump

We describe a procedure whereby structural changes that occur in muscle fibres after a rapid temperature jump can be captured by cryofixation. In the thick filament from rabbit and other mammalian skeletal muscles there is a rapid transition from a non‐helical to a helical structure as the temperature is raised from 273 K towards physiological levels. This transition is accompanied by characteristic intensity changes in the X‐ray diffraction pattern of the muscle. In our experiments to capture these changes, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump of 15–30 K from ~278 K in air. We have developed a freezing method using a modified Gatan cryosnapper in which a pair of liquid nitrogen‐cooled copper jaws were projected under pressure and closed on the fibre between 50 and 100 ms after the temperature jump. The frozen fibres were freeze‐substituted and embedded for electron microscopy. Transverse and longitudinal sections of relaxed ‘cold’ (~278 K) and temperature‐jumped fibres as well as rigor fibres were obtained. Fourier transforms of the images from the three preparations showed differences in the relative intensities of the reflections from the hexagonal filament lattice and in those of the helix‐based layer lines, similar to the differences seen by X‐ray diffraction. We conclude that we have preserved the ‘hot’ structure and that cryofixation is sufficiently fast to prevent the transition back to the ‘cold’ state.     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.