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.     

Developments of new Lowicryl® resins for embedding biological specimens at even lower temperatures

Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210K (hydrophilic K11M) and to 190K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the ‘progressive lowering of temperature’ procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo-polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.     

Low temperature embedding with Lowicryl resins: two new formulations and some applications

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.     

The control of temperature during polymerization of Lowicryl K4M: there is a low-temperature embedding method

Methods are described for controlling the temperature of Lowicryl K4M in flat embeddings and in capsules during polymerization under ultra-violet (UV) irradiation in an Agar UVF 35 low-temperature cabinet. Aluminium blocks or an ethanol bath are used as heat ‘sinks’. Consequently the question posed by Ashford et al. (1986) — “Is there a low-temperature embedding method?” — is answered in the affirmative. This control of temperature is particularly important when using gelatin capsules since a temperature rise of as little as 2 K results in uneven polymerization.     

A simple technique for in situ embedding of monolayer cultures in Lowicryl K4M

A number of difficulties are encountered in embedding monolayer cultures at low temperature in Lowicryl K4M resin. In this paper we present a simple procedure using Lab-Tek Flaskettes in which fixation, processing and embedding of monolayer cultures can readily be achieved.     

Improved sectioning and ultrastructure of bacteria and animal cells embedded in lowicryl

Lowicryl K4M-embedded Gram-positive and Gram-negative bacteria have a tendency to separate between the cell surface and the resin. This often leads to distortion of bacteria and more especially of mycobacteria. We describe attempts made to overcome this technical problem. Different assays were made on Bacillus subtilis, Escherichia coli, and Mycobacterium avium: (1) Modification of the bacterial surface by coating of bacteria with proteinic compounds; (2) treatment of bacteria with metallic salts known to modify cell wall polysaccharides; and (3) comparison between Lowicryl K4M and HM20. Conditions have been found in which the separation of all bacterial species from the resin is abolished. The most important factor appeared to be the treatment of bacteria before dehydration, with 0.5% uranyl acetate for 30 min. The second most important factor, especially for M. avium and to a lower extent for Gram-negative bacteria, was the use of Lowicryl HM20. Pre-embedding in gelatin instead of agar improved sectioning of M. avium, but had no effects on the other bacterial species. These conditions applied to macrophages infected with Shigella dysenteriae or M. avium also gave excellent results. In addition to sectioning improvement of bacteria, uranyl acetate improved the ultrastructure of bacteria and macrophages. All organelles were more clearly delineated and, hence, more easily identified. Finally, it was shown that UA treatment did not affect immunogold labeling of a variety of antigens.     

Combined quick freezing,freeze-drying,and embedding tissue at low temperature and in low viscosity resins

The techniques of quick freezing and freeze-drying provide an alternative to the more classical methodologies of chemical fixation and dehydration with organic solvents. It is possible to embed freeze-dried tissue in low viscosity resins, either at room temperature or at subzero temperatures in Spurr's resin or Lowicryl K4M, respectively. The choice of embedding medium affords additional flexibility in postdrying and embedding conditions, since Spurr's resin allows vapor fixation with osmium tetroxide and thermal polymerization. Osmium tetroxide is not recommended for Lowicryl resins, but these media permit polymerization at subzero temperatures with ultraviolet light. Both resins have unique advantages that may be utilized, depending upon the purpose of the embedding. In this paper, we discuss the details of preparing smooth muscle, from rabbit renal artery, by quick freezing and freeze-drying, as well as methods for the embedding of the freeze-dried tissue in both Spurr's resin and Lowicryl K4M. Although we have previously reported the ultrastructure of smooth muscle embedded in Spurr's low viscosity resin, the combination of freeze-drying and infiltration in Lowicryl K4M represents a new approach that allows the elimination of chemical fixation, dehydration with organic solvents, and heat polymerization of the embedding medium.     

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21°C)

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl-sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min^?1 or 2 K min^?1 were related to the distribution of the ice phase within the tissue.     

Freeze-substitution and low-temperature embedding of dairy products for transmission electron microscopy

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze‐substituted and embedded at low temperature (?20 °C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non‐dairy mixed systems were frozen by plunging in propane, at its boiling point (?187 °C). Ice cream, because it is already frozen, was fractured into 1‐mm³ pieces in liquid nitrogen and then added to frozen fixative (?196 °C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step‐wise from ?80 to ?20 °C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non‐polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at ?20 °C using ultraviolet light to cross‐link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.     

The intracellular distribution of ions and water in rat liver and heart muscle

Local dry mass concentrations of intracellular compartments in rat heart muscle and liver cells were estimated by quantitative electron microscopy and X-ray microanalysis of ultrathin frozen-dried cryosections. The results were used to calculate elemental concentrations per litre of compartment water from the X-ray microanalytical data. Water fractions were between 80.3 ± 1.3% of wet weight in the decondensed chromatin and only 45.1 ± 1.7% in mitochondria of liver cells. The lowest water fraction in heart muscle cells was also found in mitochondria. The ionic concentrations found in the cytoplasm of liver cells and in the myofibrils are in accord with the electroneutrality rule and in osmotic equilibrium with the extracellular concentrations. The concentrations of Na, K, Cl and P both in the cytoplasm and in the regions of decondensed chromatin within the nuclei were found to be equal. However, in regions of condensed chromatin K⁺ concentrations were found to be much higher than expected for a Donnan distribution of ions free in solution. Most probably the activity coefficient for K⁺ is lower in the condensed chromatin than in the decondensed or in the cytoplasm. The same holds true for the A-band as compared to the I-band in heart muscle cells. A sequestration of K⁺ was measured also in the rough endoplasmic reticulum (RER) of hepatocytes. The Cl^? concentration in mitochondria both in heart muscle and liver cells has been measured far in excess of what might be expected from a Nernstian distribution. A coupled inward Cl^? transport in mitochondria must, therefore, be assumed.     

Temperature control in Lowicryl K4M and glycol methacrylate during polymerization: is there a low-temperature embedding method?

An apparatus for embedding tissues at resin temperatures down to 228 K is described. By placing thermocouples in the resin the temperature has been monitored during embedding at low temperature with glycol methacrylate (GMA) and Lowicryl K4M. Even in this apparatus with a liquid cooling bath the heat of polymerization is not dissipated and the resin temperature rises. This rise is directly proportional to the resin temperature at the onset of polymerization and is higher in Lowicryl K4M than GMA. The initial resin temperature also affects the time taken for polymerization. The time to the onset of the peak and its duration are both increased as the temperature is lowered. This effect is more pronounced with GMA than Lowicryl K4M and polymerization of GMA is inhibited at the lowest temperature used. When Lowicryl K4M, polymerized at low temperature, is warmed up to ambient a further exothermic reaction occurs, which causes the resin temperature to rise well above ambient. Both this temperature peak and that during polymerization are reduced, but not totally eliminated, by reducing the resin volume. Aircooled systems are inefficient compared with the low-temperature apparatus used here and the resin temperature rise is consequently greater and, even with small resin volumes, it can be very high. It is therefore unlikely for published methods that the temperature specified has been maintained in the resin during polymerization. The implications of these findings are discussed in relation to enzyme and antigen survival. Recommendations include use of very small volumes of resin, refrigerated liquid-bath rather than air-cooled systems and contact with a heat sink when specimens are warmed up to ambient temperature. Examples of enzyme reaction, antigen survival and structural preservation obtained with the method are presented.     

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

Freeze-substitution of scleractinian coral for confocal scanning laser microscopy and X-ray microanalysis

Freeze-substitution in combination with confocal scanning laser microscopy provides a unique means by which the microstructure of undecalcified, and chemically untreated, polyps of scleractinian corals can be examined free of preparation artefacts. Mucocytes and intercellular spaces are particularly well preserved and the relationships of cell layers to each other and to the skeleton are undisturbed. Freeze-substitution also permits X-ray microanalysis of bulk samples and thin sections, a procedure which has hitherto been impossible to carry out on corals except on fixed tissue samples. Analyses indicated a high retention of Na⁺ and Cl^? in spaces thought to be filled with fluid similar in composition to sea water. This increases confidence in freeze-substitution as a means of retaining diffusible ions. Zooxanthellae contained metal/Ca ratios within the range of those previously reported for extracted zooxanthellae. It is shown that the mucocytes contain very high concentrations of S, K, Ca and Sr which are specifically localized in mucous granules. The concentrations differ between, and are characteristic of, different epithelial cell layers. Remarkably good correspondence was obtained between the two entirely different X-ray analytical methods. This is the first time such a comparision between methods has been made. It is suggested that the uptake of transepithelially transported Ca²⁺ and Sr²⁺ by mucocytes may be a means of regulating the deposition of these ions in the skeleton.     

Ultrastructural preservation of tissues and their reaction to the infection with trichinella in the El Plomo mummy: Muscle fiber ultrastructure and trichinosis/mummy of the Cerro El Plomo

The El Plomo mummy was a pre‐Columbian Incan child who was found mummified in the Andes Mountains above an altitude of 17,700 feet. In the environment, natural mummification occurred due to low temperatures and strong winds. Dating measurements (relative dating) by experts from the National Museum of Natural History of Chile established that the mummified body corresponds the Inca period (1,450 to 1,500 AD). In 2003, the body was transferred to the University of Chile Medical School for exhaustive medical examination. Tissue samples from the right quadriceps muscle were extracted and fixed in glutaraldehyde and postfixed in osmium tetroxide to obtain ultrathin sections to be observed by transmission electron microscope. Images were recorded on photographic paper, digitalized and analyzed by experts on morphology. Results showed a preservation of cell boundaries in striated muscle cells, but specific subcellular organelles or contractile sarcomeric units (actin and myosin) were unable to be recognized. However, the classical ultrastructural morphology of the polypeptide collagen type I was preserved intact both in primary and secondary organization. Therefore, we concluded that the process of natural mummification by freezing and strong winds is capable of damaging the ultrastructure of muscle cells and preserving collagen type I intact.     

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.     

Micrometallurgy: a technique for examining the structure of binary-element thin films over a wide range of composition

We describe a procedure for producing thin-film TEM specimens containing a thickness and/or composition gradient, utilizing an out-of-contact mask at an appropriate distance from the substrate. Imaging and diffraction capabilities of the TEM are used to examine the local structure of the film; EELS or EDX analysis provides the local elemental composition. The procedure is illustrated by results obtained from two binary-alloy systems: Se–Te (which displays a complete range of solid solubility) and Sn–Ge (where mutual solubility is very low).     

Study of potassium deficiency in cardiac muscle: quantitative X-ray microanalysis and cryotechniques

An imbalance of potassium in cardiac muscle causes an alteration of heart function. The distribution and concentration of potassium in rat papillary heart muscle was studied using cryofixation and X-ray microanalysis. Freeze-dried cryosections and sections of freeze-dried, embedded tissue were analysed. Bulk frozen specimens were freeze-dried either in a vacuum or by a new technique using liquid propane as a cryodehydration medium. These two methods of freeze-drying were tested for elemental retention in other specimens, with comparable results. A potassium concentration of 120 mmol/l was measured in normal myocytes of cardiac papillary muscle compared to 80 mmol/l in myocytes of animals stressed by a temperature of 45°C for 1 h. The presumed physiological significance of the findings is discussed.     

High-pressure freezing for the preservation of biological structure: Theory and practice

The two main advantages of cryofixation over chemical fixation methods are the simultaneous stabilization of all cellular components and the much faster rate of fixation. The main drawback pertains to the limited depth (<20 μm surface layer) to which samples can be well frozen when freezing is carried out under atmospheric conditions. High-pressure freezing increases the depth close to 0.6 mm to which samples can be frozen without the formation of structurally distorting ice crystals. This review discusses the theory of high-pressure freezing, the design of the first commercial high-pressure freezing apparatus (the Balzers HPM 010), the operation of this instrument, the quality of freezing, and novel structural observations made on high-pressure-frozen cells and tissues.     

Suggestions for the quantitative X-ray microanalysis of thin sections of frozen-dried and embedded biological tissues

When a microregion in a thin section of frozen-dried and embedded tissue is analysed by the conventional electron-probe X-ray continuum-normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a ‘tag’ element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measurable, and the X-ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze-drying or by incomplete replacement of water by embedding medium. The same X-ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry-mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.     

Quantitative microlocalization of diffusible ions in normal and galactose cataractous rat lens by secondary ion mass spectrometry

Secondary ion mass spectrometry (SIMS) is a surface analytical technique with high sensitivity for elemental detection and microlocalization capabilities within the micrometre range. Quantitative analysis of epoxy resins and gelatin have been reported (Burns-Bellhorn & File, 1979).