Scanning microscopy. Supplement最新文献

The three-dimensional visualization of the 400 micron thick, transparent, in situ cornea is described to demonstrate the use of confocal light microscopy for noninvasive imaging of living cells and thick tissues in their normal, vital conditions. Specimen preparation and physiological stability, as well as light attenuation corrections are critical to data acquisition. The technique to provide mechanical stability of the specimen during the duration of the image acquisition is explained. A laser scanning confocal light microscope (LSCM) was used to obtain optical serial sections from rabbit eyes that were freshly removed and placed in a physiological Ringer's solution. This study demonstrates the capability of the confocal light microscope to obtain a series of high contrast images, with a depth resolution of one micron, across the full thickness of living, transparent tissue. The problems of nonisotropic sampling and the limited eight-bit dynamic range are discussed. The three-dimensional reconstructions were obtained by computer graphics using the volume visualization projection technique. The three-dimensional visualization of the cornea in the in situ eye is presented as an example of image understanding of thick, viable biological cells and tissues. Finally, the criterion of image fidelity is explained. The techniques of confocal light microscopy with its enhanced lateral and axial resolution, improved image contrast, and volume visualization provides microscopists with new techniques for the observation of vital cells and tissues, both in vivo and in vitro.

Cryo-electron microscopy of single biological particles poses new challenges to digital image processing due to the low signal-to-noise ratio of the data. New tools have been devised to deal with important aspects of 3-D reconstruction following the random-conical data collection scheme: (a) a new shift-invariant function has been derived, which promises to facilitate alignment and classification of single particle projections; (b) a new method of orientation search is proposed, which makes it possible to relate random-conical data sets to one another prior to reconstruction; and (c) the foundation is laid for a 3-D variance estimation which utilizes the oversampling of 3-D angular space by projections in the random-conical reconstruction scheme.

Three-dimensional structures have recently been determined by electron crystallography at a resolution high enough to determine atomic arrangements in both protein and mineral specimens. The different nature of these two types of specimens produces some very significant differences in the way data is obtained and processed, although the principles are the same. The sensitivity of proteins to damage by the electron beam limits the signal-to-noise ratio in the image and the resolution to which data can be extracted from the image. A number of constraints, such as the amino acid sequence and the connectivity of atoms within amino acids, can be used in interpreting the limited image data. In materials samples, the relative insensitivity to damage allows obtaining resolution limited only by the microscope. In many samples, dynamical scattering and other non-linear effects limit the information in the image, but this limit can be circumvented by working in very thin areas of the specimen.

Hexagonal ice crystals formed in frozen biological specimens are large and branched. They can produce severe structural damage by solute segregation but there are also cases where they seem to cause only minor damage. When cooling is more rapid, cubic ice crystals can be formed. These are small and in general, they cause little damage. These observations can be readily explained with the hypothesis that large hexagonal ice crystals can originate from the rewarming induced transformation of a large number of cubic ice crystals. This transformation would take place without significant solute displacement.

A number of diseases is associated with changes in ion and/or water distribution at the tissue or cell level, and X-ray microanalysis can be used to investigate the relationship between the disease process and the changes in elemental distribution. For analysis of diffusible elements by X-ray microanalysis, the tissue has to be prepared by cryotechniques. To carry out studies of this kind in a clinical environment poses a number of problems. Some of these problems occur already before the tissue is frozen, others are caused by the practical and ethical limitations that are imposed on the freezing method itself when human tissue is to be used. The use of cryostat sections for analysis at the cellular level, and of in vitro systems and cell cultures in which sampling and cryopreparation can be separated in time and place can be useful alternatives.

Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies. These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias). The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates. Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels. Improved procedures were developed to produce the polystyrene films of required qualities. These films allowed processing of cells for electron microscopy including chemical fixation, cryo-immobilization, and immunolabelling. Furthermore, these polystyrene films permitted observations of the same cell in the high voltage electron microscope to reveal the internal organization and in the low voltage scanning electron microscope to reveal the surface topography. Neoplastic cells in suspension may dramatically change their ultrastructure as a result of interactions with substrates or other cells. Therefore, immobilization of cellular processes must occur rapidly while cells remain in suspension. These processes were cryo-immobilized by high pressure freezing through the use of the newly designed specimen carrier. Procedures allowing high yield attachment of cryo-fixed neoplastic cells to amino-propyl-derived glass carriers enabled observations of cell surface topography. Furthermore, freeze-substitution and drying of freeze-fractured cells revealed their three-dimensional internal organization in the low voltage scanning electron microscope.

A method is presented for obtaining simple approximate solutions for the problem of self-diffusion in an ordered array of obstacles. Our results are compared with some previous exact and approximate solutions, and we find that our method agrees well with the exact results over a large range of the volume fraction of the obstructions. It is shown that there is an important distinction between measurements of the diffusion coefficient by the capillary flow method and the spin-echo method. The modifications for the spin-echo case are given and applied to recent measurements on the anisotropy of the self-diffusion of water in striated muscle and to measurements on cysts of the brine shrimp. The analysis shows that very large volume fractions of obstructive barriers are required in order to account for the reduction in the diffusion coefficient in biological systems. Thus this model analysis leads to the supposition that a substantial fraction (20-40%) of the cell water is hydration water, or that the diffusion coefficient of the cytoplasmic water is reduced substantially from the free water value. In either case, the conclusion that a substantial fraction of cell water has diffusive properties that are altered by the macromolecules of the cytoplasm seems inescapable. In the case of NMR methodology, the measuring times are such that the values for diffusion are often influenced by the presence of macromolecular structures (obstructions) within the cells. This suggests that obstructions make a significant contribution to the value of the NMR diffusion coefficient and that NMR may have practical value for the evaluation of obstruction effects.

Despite a plethora of reports on the ultrastructure of secretory granule release by exocytosis, the release of coagulant activity from stimulated platelets is still being attributed to membrane vesiculation. Membrane vesiculation and the formation of myelin figures have been shown to be artifacts of glutaraldehyde GA fixation. Cells fixed by direct osmium or rapid freezing are free of such structures. Yet there is still doubt that rapid freezing interferes with vesiculation process. This study has addressed this issue by examining: (1) whether freezing and freeze-substitution affects membrane vesiculation, (2) whether paraformaldehyde-fixation also induces the phenomenon, and (3) whether the aldehyde concentration is of influence. Aldehyde fixation was carried out prior to impact freezing and freeze-substitution. In thrombin-stimulated platelets, membrane vesiculation and myelin figures were found. Glutaraldehyde induced multivesicular structures, paraformaldehyde or low aldehyde concentrations only blebs on the platelet surface. The membrane vesicles were in continuity with the cytoplasmic matrix. Unstimulated platelets did not show vesiculation or myelin figures. Control samples, without aldehyde fixation, showed instead of membrane vesiculation, granule fusion with the plasmalemma, or, instead of myelin figures, compound granules. This confirms that membrane vesiculation and the formation of myelin figures are artifacts induced by the failure of aldehydes to arrest lipid mobility within membranes undergoing rapid changes in structure. Although the presence of membrane vesiculation and myelin figures in platelets indicates that exocytotic processes were occurring at the moment of aldehyde fixation, the finding of membrane vesiculation in aldehyde-fixed platelets does not indicate a separate type of exocytosis.

A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs(+)-ions which are known to accumulate in living cells like K(+)-ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be stained with electron-dense Cs(+)-ions: protein sites of preferential ion adsorption are visualized. These sites are similar to those accumulating monovalent ions in living cells as had been shown previously with frozen-hydrated preparations. An observed pH-dependency of the adsorption staining is consistent with the view that the ion adsorption sites are beta- and gamma-carboxyl groups of cellular proteins. The results obtained so far indicate that the new method can be used to investigate weak interactions between cellular proteins and different ions by electron microscopic methods.

The success of post-embedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level. In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and are very difficult to preserve in ultrathin cryosections. Lowicryl sections of freeze-substituted lung tissue shows excellent preservation of lamellar bodies in combination with immunogold localization of a hydrophobic surfactant protein. With an antibody against the Forssman glycolipid we demonstrate a highly reproducible intracellular localization of this glycolipid with high specificity and resolution. This method results in the retention of lipids and glycolipids and allows postembedding immunogold labeling.