Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material

This article presents the best current practices for preparation of biological samples for examination as thin sections in an electron microscope. The historical development of fixation, dehydration, and embedding procedures for biological materials are reviewed for both conventional and low temperature methods. Conventional procedures for processing cells and tissues are usually done over days and often produce distortions, extractions, and other artifacts that are not acceptable for today’s structural biology standards. High-pressure freezing and freeze substitution can minimize some of these artifacts. New methods that reduce the times for freeze substitution and resin embedding to a few hours are discussed as well as a new rapid room temperature method for preparing cells for on-section immunolabeling without the use of aldehyde fixatives.     

A comparison of cryo- versus chemical fixation in the soil green algae Jaagiella

Chemical fixation protocols provided unsatisfactory preserved material for ultrastructural studies on Jaagiella alpicola Vischer (Chlorophyta). Instead, several methods of rapid freeze fixation followed by freeze substitution were applied. For fast freeze fixation, two methods were tried: plunge immersion freezing in liquid propane using a home-made device, and projection against a copper block cooled by either liquid nitrogen or liquid helium. Each method furnished well fixed material. The quality of the fixed samples was quite similar whether propane or the cryoblock cooled with liquid nitrogen was used. Liquid helium, however, provided superior results. After fixation the samples were cryosubstituted, using acetone or methanol as organic solvent with a chemical fixative added. Acetone gave better results than methanol as a substitution solvent when high temperature embedding was performed. The best cryosubstitution for ultrastructural studies was that in which osmium tetroxide or a mixture of osmium tetroxide and urany acetate was used.     

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes

Summary— Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes; ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.     

Synthesis of starch derivatives with labile cationic groups

A new route to starch derivatives bearing hydrolyzable cationic groups was developed. This was based on reacting starch compounds with betaine derivatives in the presence of diisopropylcarbodiimide and 4-dimethylaminopyridine as coupling reagents in an aprotic polar solvent. Water-soluble starches with a perfectly controlled degree of substitution were thus obtained which were fully characterized by infrared, ¹H and ¹³C spectroscopy and viscosity measurements. The cationic groups grafted on the polysaccharides are shown to hydrolyze slowly upon storage at room temperature.     

Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses

In order to develop successful cryopreservation protocols a better understanding of the freeze- and dehydration-induced changes occurring in the cell membrane and its underlying support, the actin cytoskeleton, is required. In this study, we compared the biophysical response of model mammalian cells (human foreskin fibroblasts) to hyperosmotic stress and freeze/thaw. Transmitted light, infrared spectroscopy, fluorescence- and cryo-microscopy were used to investigate the changes in the cell membrane and the actin cytoskeleton. We observed that a purely hyperosmotic challenge at room temperature resulted in bleb formation. A decrease in temperature abrogated the blebbing behavior, but was accompanied by a decrease in viability. These results suggested that cell survival depended on the availability of the membrane material to accommodate the volumetric expansion back to the original cell volume at isotonic conditions. Our data also showed that freeze/thaw stresses altered the cell membrane morphology resulting in a loss of membrane material. There was also a significantly lower incidence of blebbing after freeze/thaw as compared to isothermal osmotic stress experiments at room temperature. Significant depolymerization of the actin cytoskeleton was seen in cells whose membranes had been compromised by freeze/thaw stresses. Actin depolymerization using cytochalasin D affected the stability of the membrane against mechanical stress at isothermal conditions. This study shows that both the membrane and cytoskeleton, as a system, are involved in the osmotic and freeze/thaw-induced responses of the mammalian cells.     

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.     

Regioselective silylation of N-phthaloylchitosan with TBDMS and TBDPS groups

N-Phthaloylchitosan represents a key intermediate for the regioselective modification of chitosan in organic media. Chemoselective protection of primary alcohols on N-phthaloylchitosan was achieved with tert-butyldimethylsilyl (TBDMS) and tert-butyldiphenylsilyl (TBDPS) groups in imidazole/DMF and DMAP/pyridine. Influence of experimental conditions such as solvent, choice of base, stoichiometry, temperature, and time of reaction was studied regarding the degree of substitution (ds) of silyl groups. TBDMS groups allow higher ds than TBDPS groups. Higher reaction temperatures in different conditions led to lower ds and incomplete substitution. However, regioselective silylation of N-phthaloylchitosan was realized with ds up to 0.92 at room temperature. Silylated derivatives were characterized by elemental analysis, IR, and CP/MAS 13C NMR spectroscopies.     

Freeze drying and freeze substitution combined with low temperature-embedding

Summary An X-ray microanalytical and morphological investigation was carried out on rapidly frozen freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze substitution and freeze drying. The investigation also included an analysis of specimens which had been infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl-HM20. The method of freeze drying, followed by embedding and polymerisation at low temperatures in vacuo was found to give satisfactory results, comparable with more tedious and hazardous freeze substitution technique.     

不同干燥方式、存储温度对羊栖菜中岩藻黄素稳定性的影响

为探究不同干燥方式、不同存储温度和存储时间对羊栖菜中岩藻黄素稳定性的影响,本研究考察了三种不同干燥方式,包括自然阴干、低温烘干和冷冻干燥,对羊栖菜中岩藻黄素稳定性的影响。结果表明采用冷冻干燥所得羊栖菜,含水量低,岩藻黄素含量高,达到699.2μg/g,岩藻黄素保留率为94.7%,远高于自然阴干及低温烘干。因此,冷冻干燥是最佳干燥方法。考察了四个不同温度(-20、4、25、30℃)存储对冻干羊栖菜中岩藻黄素稳定性的影响,结果表明随着存储温度的升高,岩藻黄素的降解率明显增高,因此,-20℃为最佳储存温度。     

Freeze fracture evidence for lateral phase separations in the plasmalemma of chilling-injured avocado fruit

Summary Unripe avocado fruit (Persea americana Mill. cv Hass) were held at 6 °C either in air or in an atmosphere with 100 PPM ethylene and were assessed for chilling injury after one and two weeks. Injury did not occur in any fruit after one week. After two weeks, the fruit in air were still uninjured, but the fruit subjected to ethylene exhibited chilling injury. When the uninjured fruit (both air-treated for one and two weeks and ethylene-treated for one week) were allowed to warm to room temperature before freezing for freeze fracture electron microscopy, replicas revealed membranes with a randomly dispersed pattern of intramembranous particles (IMPs). However, when these uninjured fruit were frozen for freeze fracture without warming, particle-free domains were visible in the plasmalemma. The membranes of the ethylene-treated, chilling-injured (2 weeks) fruit, on the other hand, contained particle-depleted regions in the plasmalemma of fruit frozen not only from 6 °C but also in those allowed to warm to room temperature before freezing for freeze fracture. These particle depleted microdomains were not seen in fruit kept continuously at room temperature (20 °C), even in the presence of high levels of endogenous ethylene which is produced during normal ripening. We suggest these particle-depleted microdomains formed in the fruit frozen for freeze fracture from low temperatures and in the chilling-injured fruit to be due to lateral phase separations of the membrane components, possibly due to an increase in the viscosity of some membrane lipids, leading to the formation of microdomains of gel phase lipid in the plane of the membrane. These phase separations appear to be initially reversible by raising the temperature, however, this reversibility is apparently lost after injury has occurred. With regard to the cause of chilling injury in avocados, we suggest that some secondary effect is involved due to the long term presence of gel phase lipids in the membrane.     

Formation and stability of oxygen-rich bubbles that shape photosynthetic mats

Gas release in photic-zone microbialites can lead to preservable morphological biosignatures. Here, we investigate the formation and stability of oxygen-rich bubbles enmeshed by filamentous cyanobacteria. Sub-millimetric and millimetric bubbles can be stable for weeks and even months. During this time, lithifying organic-rich laminae surrounding the bubbles can preserve the shape of bubbles. Cm-scale unstable bubbles support the growth of centimetric tubular towers with distinctly laminated mineralized walls. In environments that enable high photosynthetic rates, only small stable bubbles will be enclosed by a dense microbial mesh, while in deep waters extensive microbial mesh will cover even larger photosynthetic bubbles, increasing their preservation potential. Stable photosynthetic bubbles may be preserved as sub-millimeter and millimeter-diameter features with nearly circular cross-sections in the crests of some Proterozoic conical stromatolites, while centrimetric tubes formed around unstable bubbles provide a model for the formation of tubular carbonate microbialites that are not markedly depleted in ¹³C.     

Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at ?80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at ?20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC–ESI–MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC–ESI–MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC–ESI–MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at ? 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at ? 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at ? 80 °C, LN2, FDRT or FD-20 °C for up to a year.     

Compositional Discrimination of Decompression and Decomposition Gas Bubbles in Bycaught Seals and Dolphins

Gas bubbles in marine mammals entangled and drowned in gillnets have been previously described by computed tomography, gross examination and histopathology. The absence of bacteria or autolytic changes in the tissues of those animals suggested that the gas was produced peri- or post-mortem by a fast decompression, probably by quickly hauling animals entangled in the net at depth to the surface. Gas composition analysis and gas scoring are two new diagnostic tools available to distinguish gas embolisms from putrefaction gases. With this goal, these methods have been successfully applied to pathological studies of marine mammals. In this study, we characterized the flux and composition of the gas bubbles from bycaught marine mammals in anchored sink gillnets and bottom otter trawls. We compared these data with marine mammals stranded on Cape Cod, MA, USA. Fresh animals or with moderate decomposition (decomposition scores of 2 and 3) were prioritized. Results showed that bycaught animals presented with significantly higher gas scores than stranded animals. Gas composition analyses indicate that gas was formed by decompression, confirming the decompression hypothesis.     

Freeze substitution of fungi for cytological analysis

The preparatory techniques of freeze substitution for electron microscopy are given in detail. Included is a simple, reliable method for the selection of individual freeze-substituted cells from flat embedments via light microscopy for subsequent electron microscope analysis. These mthods are intended for studying cells in monolayer cultures, tissues, suspensions of cells, or cell fractions and are widely applicable for cell biologists in any field. An historical background and review of the relevant literature is included so that the reader will be fully prepared to use of modify the techniques and to troubleshoot results as required. All materials that are needed are thoroughly discussed so that any laboratory can adopt freeze substitution as a routine procedure.     

Effect of Postmortem Factors on Muscarinic Receptor Subtypes in Rat Brain

Although prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4 degrees C and -20 degrees C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]Pir). We found that delay at room temperature up to 4 h, or storage at 4 degrees C for 24 h or at -20 degrees C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding.     

Release of Volatile Mercury from Vascular Plants

Volatile, organic solvent soluble mercury has been found in leaves and seeds of several angiosperms. Leaves of garlic vine (Pseudocallyma alliacium), avocado (Persea americana) and haole-koa (Leucaena glauca) release mercury in volatile form rapidly at room temperature. In garlic vine, the most active release is temperature dependent, but does not parallel the vaporpressure temperature relationship for mercury. Mercury can be trapped in nitric-perchloric acid digestion fluid, or n-hexane, but is lost from the hexane unless the acid mixture is present. Seeds of haole-koa also contain extractable mercury but volatility declines in the series n-hexane (90%) > methanol (50%) > water (10%). This suggests that reduced volatility may accompany solvolysis in the more polar media. Gas chromatographic analysis shows that the volatile compound is not dimethyl mercury.     

Photobactericidal plastic films based on cellulose esterified by chloroacetate and a cationic porphyrin

The synthesis and characterisation of pyridinium porphyrinic chloroacetyl cellulose ester chlorides, where photosensitizing agents are covalently bounded to the polymeric chain, is presented in this paper. First, cellulose was homogenously converted into chloroacetate cellulose ester in DMAc/LiCl solvent by using chloroacetyl chloride. The complete substitution of cellulose was achieved using 7equiv of chloroacetyl chloride for a 2h reaction at room temperature. The absence of base did not prove detrimental to reaction. The grafting of monopyridyltritolylporphyrin onto chloroacetate cellulose ester was then realised by alkylation of the photosensitizer in DMF. These new plastic films were found to be thermostable up to 55 degrees C; higher temperatures led to progressive deacetylation. First results of their photobactericidal activity against Staphylococcus aureus and Escherichia coli strains are very encouraging. Such materials could find applications in medical environments as an alternative to overcome the rampant bacterial multiresistance to classical antibiotics.     

The ultrasonic degradation of biological macromolecules under conditions of stable cavitation. II. Degradation of deoxyribonucleic acid

Solutions of T7 bacteriophage or calf thymus DNA arc degraded in solution by ultrasonic fields of low intensity in the presence of vibrating air bubbles but are not degraded at these low intensities when such bubbles are absent. Evidence is presented for the hydrodynamic nature of the observed degradation and theoretical simulation of a plausible degradation mechanism is compared with experimental degradation studies. It is concluded that degradation of such linear macromolecules as DNA may occur as a result of stresses induced in the macromolecule; these stresses are the result of a relative movement of solvent molecules and the macromolecules in the time-independent flow of solvent near the vibrating bubbles.     

The basement membranes of cryofixed or aldehyde-fixed,freeze-substituted tissues are composed of a lamina densa and do not contain a lamina lucida

When tissues are processed for electron microscopy by conventional methods, such as glutaraldehyde fixation followed by rapid dehydration in acetone, basement membranes show two main layers: the electron-lucent lamina lucida (or rara) and the electrondense lamina densa. In an attempt to determine whether this subdivision is real or artefactual, two approaches have been used. Firstly, rat and mouse seminiferous tubules, mouse epididymis and associated tissues, and anterior parts of mouse eyes were subjected to cryofixation by instant freezing followed by freeze substitution in a-80° C solution of osmium tetroxide in dry acetone, which was gradually warmed to room temperature over a 3-day period. The results indicate that, in areas devoid of ice crystals, basement membranes consist of a lamina densa in direct contact with the plasmalemma of the associated cells without an intervening lamina lucida. Secondly, a series of tissues from mice perfused with 3% glutaraldehyde were cryoprotected in 30% glycerol, frozen in Freon 22 and subjected to a 3-day freeze substitution in osmium tetroxide-acetone as above. Under these conditions, no lamina lucida accompanies the lamina densa in the basement membranes of the majority of tissues, including kidney, thyroid gland, smooth and skeletal muscle, ciliary body, seminiferous tubules, epididymis and capillary endothelium. Thus, even though these tissues have been fixed in glutaraldehyde, no lamina lucida appears when they are slowly dehydrated by freeze substitution. It is concluded that the occurrence of this lamina in conventionally processed tissues is not due to fixation but to the rapid dehydration. However, in this series of experiments, the basement membranes of trachea and plantar epidermis include a lamina lucida along their entire length, while those of esophagus and vas deferens may or may not include a lamina lucida. To find out if the lamina lucida appearing under these conditions is a real structure or an artefact, the trachea and epidermis were fixed in paraformaldehyde and slowly dehydrated by freeze substitution. Under these conditions, no lamina lucida was found. Since this result is the same as observed in other tissues by the previous approaches, it is proposed that the lamina lucida is an artefact in these as in the other investigated basement membranes. Thus, basement membranes are simply composed of a lamina densa that closely follows the plasmalemma of the associated cells. At high magnification, the lamina densa consists of a tridimensional network of cords, while the plasmalemma is covered by a glycocalyx; close contact is observed between cords and glycocalyx and is interpreted by assuming that the laminin present in the cords binds to laminin receptors in the glycocalyx.     

The effect of freeze/thaw cycles on the stability of compounds in DMSO

A diverse set of 320 compounds from the Procter & Gamble Pharmaceuticals organic compound repository was prepared as 20-mM DMSO solutions and stored at 4 degrees C under argon in pressurized canisters to simulate a low-humidity environment. The plates were subjected to 25 freeze/thaw cycles while being exposed to ambient atmospheric conditions after each thaw to simulate the time and manner by which compound plates are exposed to the atmosphere during typical liquid-handling and high-throughput screening processes. High-performance liquid chromatography-mass spectrometry with evaporative light-scattering detection was used to quantitate the amount of compound remaining after every 5th freeze/thaw cycle. Control plates were stored either at room temperature under argon or at 4 degrees C under argon without freeze/thaw cycling and were evaluated at the midpoint and the endpoint of the study. The study was conducted over a short time period (i.e., 7 weeks) to minimize the effect of compound degradation over time due to the exposure of the compounds to DMSO. The results from this study will be used to determine the maximum number of freeze/thaw cycles that can be achieved while maintaining acceptable compound integrity.