Scanning microscopy. Supplement最新文献

Many techniques for processing tissue into resin are available, varying from conventional room temperature to low temperature procedures. The problem is to choose an appropriate method to suit the biological specimen under study. Room temperature approaches with aldehyde and osmium fixation do not give optimal retention of immunoreactivity. Osmium can be removed from sections, but recovery of immunosensitivity is reduced. Osmium post-fixation can be omitted, but heat polymerization of resins causes tissue extraction and loss of immunoreactivity. Alternative techniques rely on the use of milder polymerization methods and avoid osmium. However, while providing an improvement, this alone is not sufficient to maximize tissue reactivity. Fixation with high concentrations of glutaraldehyde (greater than 1%) and processing into resin at either room or low temperature results in retention of similar levels of immunoreactivity. Low concentration glutaraldehyde (less than 0.2%) fixation for short periods of time (less than 60 minutes) produces improved tissue immunoreactivity and allows low concentrations of antigen at secondary sites to be detected. However, the tissue is now only minimally stabilized and is prone to extraction and conformational damage during processing. It can be partially protected by employing one of two strategies: processing at room temperature with partial dehydration (upto 70% solvent) and rapid embedding in LR White or Lowicryl K4M at 0 degrees C, or processing at progressively lower temperatures (PLT) and embedding in Lowicryl at -35/-50 degrees C. In a third strategy, specimens sensitive to very low fixative concentrations are cryo-immobilized, then resin embedded after substitution or freeze-drying (this latter method awaiting evaluation for inclusion in our strategical approach).

Chemically fixed pancreas was infiltrated with various cryoprotectants to obtain homogeneously vitrified samples upon cooling. The suitability of these samples for cryoultramicrotomy was tested. Contrast was hardly detectable initially in thin cryo-sections but increased upon irradiation, irrespective of the cryoprotectant (glycerol, propylene glycol, methanol) used. Contrast and beam damage were analyzed in vitrified thin films from collagen, phospholipid vesicles and various concentrations of glycerol. Glycerol increased the beam sensitivity of both collagen and phospholipid vesicles, but diminished the contrast between matrix and lipid vesicles or collagen fibers. The effects of glycerol as observed in thin films explain some of the effects of cryoprotectants in thin cryo-sections. To reduce beam damage in vitrified specimens two approaches are proposed. Firstly, when vitrified films are prepared, dilute suspensions should be used without cryoprotectant. In some cases, such as (thin) intact cells, the composition of the suspended material can only be marginally influenced. Then a second approach can be used involving the application of higher accelerating voltages (e.g. 300 kV). This has two advantages; the increase in mean free path-length of the electrons causes less beam damage on one hand and allows better resolution of thick specimens on the other hand. Micrographs from E. coli bacteria vitrified from suspension illustrate some of the potentials of "intermediate voltage" cryo-electron microscopy.

X-ray microanalysis can be an important tool to reveal the spatial relationships between polyelectrolytes, ions, and water as they occur within cells and tissues in vivo. To reach this goal, at least two of these three closely interrelated variables should be measured independently. Moreover, the absence of systematic errors should be proven. The present review discusses the probability of artificial ion and water shifts between intracellular compartments due to the growth of dendritic ice crystals much larger than the cross-sectioned remnants commonly seen in frozen-dried sections. Considering the possible mechanism of ice crystal growth it is concluded that ions and water are not translocated over large distances. Moreover, problems associated with the preparation of a sample for water content estimations are discussed here. The importance of an appropriate pre-freezing treatment is highlighted, as is the importance of fast freezing. The risk of artificial water shifts between compartments with different freezing properties is discussed and the absence of clefts between compartments or haloes around them as seen in frozen-dried sections is taken as an appropriate criterion. Constancy of section thickness and retention of full hydration of cryosections are necessary prerequisites for many of the techniques and conditions to fulfill these requirements are given.

The papers presented at this Pfefferkorn Conference demonstrate the dramatic recent progress in the science of biological specimen preparation for electron microscopy. This progress results largely from increased use of more diverse, critical, correlative scientific methods. This paper outlines several strategies that tend to promote this type of scientific approach, and that have proven generally useful in biological research. The strategy most commonly chosen to augment both the empirical and the cross-disciplinary components of structural studies is the correlative use of diverse experimental techniques on samples which are parallel to those prepared for microscopy. This type of approach tends to advance our understanding of biological structure and function and also of the scientific methodology. Such approaches redirect attention to the biological problem under study and tend to open new areas of investigation. A second strategy which promotes more rigorous scientific approaches is the application of correlative techniques to identical structures. In contrast with parallel studies, data from identical structures document directly the coincidence of different features within each individual structure, and these data establish the distributions of these features in the study population based on relatively few observations. A third strategy to promote more critical science is to utilize the effects of the specimen preparation as experimental parameters by varying the preparative methods with appropriate controls. This approach is especially valuable in studies of biological specimen preparation, where the potential impact of systematic errors warrants especially rigorous scientific practice.

Dried biological samples are low in scattering power, non-conducting and sensitive to radiation damage. These facts complicate the choice of the optimum beam voltage Vo at which they should be observed in the scanning electron microscope (SEM) because they add as variables the type and thickness of the coating material and degradation/contamination of the specimen by the beam. Heretofore, high resolution SEM could only be carried out at relatively high Vo (20-30kV) because available equipment could not produce small beam diameters at low Vo. Modern instruments can produce beam diameters of about 3nm at 1.5kV. As normal preparative procedures (fixation, critical point drying, coating) are unlikely to preserve reliable structure below this level, it is now possible to investigate the possible advantages associated with low Vo operation such as a reduction in charging and radiation damage and improved topographic contrast. The conclusion recommended by this paper is that the term resolution needs careful definition. The size of the smallest features visible in a micrograph is a function of many variables. Although probably the most important is specimen preparation, a number of others (probe size, beam penetration range, contamination, coating thickness needed to provide contrast and avoid charging etc) are functions of Vo. Of these variables at least probe size and possibly contamination become more favorable at higher Vo while the remainder favor low Vo. As a result the optimum will occur at a Vo where the best balance of these factors occurs for a particular sample. When using the Hitachi S-900, we have found that the optimum seems to be at 1.5-2.5kV for topologically diverse samples, but may extend to 5kV on samples on which very small structural details have been preserved and which are relatively stable to radiation damage.

Initially developed for the in situ localization of nucleic acids, the enzyme-gold approach has been extended to the detection of a large variety of biological molecules. The enzyme-gold approach, based on the highly specific interaction existing between an enzyme and its substrate, can be used both in pre-embedding and post-embedding labeling procedures. Fixation and embedding conditions for the best preservation of each particular substrate under study have to be defined. On the other hand, conditions required to adsorb purified enzymes on colloidal gold particles should be determined according to the biochemical properties of each protein. Labeling protocols must be performed taking into consideration the optimal conditions for the enzymatic activity. The enzyme-gold complexes have been shown to retain their biochemical properties and the specificity of each labeling obtained has been assessed through various control experiments. Initially applied for the demonstration of nucleic acids, the approach has been extended to the ultrastructural localization of various substrates, and in particular, more recently, glycoconjugates and phospholipids. Indeed, various glycosidase-gold complexes and a phospholipase-gold complex, applied in pre- and post-embedding labeling protocols, did specifically label plasma membranes as well as various defined subcellular compartments. In addition, the morphometrical evaluation of labeling intensities revealed differences in amounts of binding sites between compartments. Considering its versatility, simplicity and efficiency, the enzyme-gold technique provides an alternative, very valuable cytochemical tool for the localization of a variety of biological molecules at the cellular and subcellular level.

Video-enhanced interference reflection microscopy (VEIRM) was used to examine contact sites between the ventral membrane of intact platelets and underlying surfaces. It was observed that the ventral membrane in the central granulomere region of fully spread platelets was separated from the surface, while the membrane in other regions was mostly in close contact. The VEIRM image of intact platelets was compared with the video-intensified fluorescence microscopic (VIFM) image of cytoskeletal structures labeled with rhodamine-phalloidin. The VEIRM was also used to visualize the cytoskeletal structures of platelets spread on glass surfaces. Platelets were treated with Triton X-100, glutaraldehyde and acetic acid in sequence. This method was used to follow the sequence of cytoskeletal reorganization of platelets after surface-induced activation. In addition, the effects of albumin and fibrinogen on the cytoskeletal reorganization of spreading platelets were investigated. On fibrinogen-coated surfaces, platelets developed extensive inner filamentous zones which encircled the central granulomere region. In the presence of albumin, however, platelet inner filamentous zones were very poorly developed.

Osmium impregnation techniques have become useful for imparting conductivity to tissue specimens for SEM, thereby avoiding coating with gold or other metals. Such techniques have been developed to produce aesthetically pleasing images of mammalian (particularly human) chromosomes prepared by standard cytogenetical methods which use methanol-acetic acid fixation. The present study was designed: (1) to examine changes in the appearance of chromosomes as a result of preparation by osmium impregnation techniques; (2) to assess the function and importance of the various stages of chromosome preparation; and (3) to identify the chemical groups responsible for osmium binding. Methanol-acetic acid fixed chromosomes are known to have lost many proteins during fixation, and appear to be flattened down on the substrate. Osmium impregnation swells these flattened chromosomes to a variable extent, but the result is inevitably an artefact, albeit a useful one, and not a true representation of the chromosome in vivo. The size of chromatin fibres, for example, is the consequence of the degree of protein extraction during fixation, the loss of material during pre-treatments (e.g. trypsin), and the amount of osmium uptake during impregnation. Trypsin pre-treatment removes a surface coating of protein from the chromosomes as well as exposing chemical groups which can react with osmium. The principal reactive site appears to be amino groups, which bind glutaraldehyde, which in turn binds thiocarbohydrazide, to which the osmium becomes attached. Pre-treatments other than trypsin can be used to extract chromosomal material and to reveal different aspects of chromosome structure.

Borrelia burgdorferi--the Lyme disease spirochete--was grown in modified Kelly medium and characterized by transmission and by scanning electron microscopy. Using silver staining procedures which preferentially bind to nuclear components of eukaryotic cells, signal could be detected by backscattered electron imaging throughout the length of the prokaryotic spirochete. Interestingly, however, the highest levels of backscattered signal were observed in naturally elaborated membrane blebs that were visible attached to cell surfaces and free in the medium. These membrane vesicles could be enriched by filtration through nitrocellulose or Anopore membranes and by differential centrifugation. The possibility of contaminating cellular DNA coating the membrane vesicles was ruled out by exhaustive digestion with pancreatic DNAse I. Intact DNA was demonstrated both by lysing blebs directly on the surface of microscope grids and by extracting molecules from purified bleb preparation with detergents and solvents. Both linear and circular DNA molecules could be identified in purified membrane blebs. A simple, one-step, alternative silver staining procedure is described which appears to effectively label the protein-nucleic acid complexes contained in the membrane vesicles of the human pathogen B. burgdorferi, and may provide an important method to track and to define the biological function of these structures.