Scanning microscopy. Supplement最新文献

Cryoelectron microscopy allows the observation of hydrated samples at high spatial resolution, and it would be of great interest in biology to apply this method to cells and tissues. However, because of technical problems, the cryo-observation of frozen hydrated ultrathin sections of bulk material has not become an established method. The major limitations are due to the difficulty of achieving the vitrification of such material, and the structural deformation caused by ultrathin sectioning: 1. The vitrification of cells in a physiological environment requires high-pressure freezing. However, new results suggest that the pressure may alter the ultrastructure of the sample. 2. Cryosectioning compresses structures in the cutting direction about 40%. This deformation does not necessarily destroy the character of macromolecular assemblies, but since it depends on the properties of the material, internal standards cannot be used to correct for the deformation of all the structures in a cell.

Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells. Fixation of plant tissues to preserve cytoplasmic structure poses special problems. When possible, emphasis should be placed on the imaging of structures in stained living cells over time. The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging. The first observable events after Nod factor stimulation occur in root hairs and are changes in rates of cytoplasmic streaming, nuclear movements, and changes in the shape of the vacuole. Within ten minutes, the endoplasmic reticulum shifts position towards the tip of the root hair. For comparison, the endoplasmic reticulum localization in pollen tubes and onion epithelial cells will be illustrated. The actin cytoskeleton undergoes a series of changes over a twelve hour period. These changes in the cytoskeleton are spatially and temporally correlated with the observed growth changes of the root hairs. This dynamic change of the actin filament and endoplasmic reticulum and associated secretory vesicles in these root hairs suggests a mechanism for the observed root hair growth changes.

Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the current approach to sample preparation. We are working on approaches based on chemical functionalization of mica. Treatment of mica with aminopropyltriethoxy silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water. We have shown that AP-mica is an appropriate substrate for numerous nucleoprotein complexes as well. The AFM images of the complex of DNA with RecA protein are stable and indicate a structural periodicity for this filament. AP-mica holds strongly such large DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrate for their imaging with AFM. We have further develop this approach for making hydrophobic substrates. Silylation of mica surface with hexamethyldisilazane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna complex isolated from green photosynthetic bacteria. Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide. These activated surfaces show high activity towards binding the DNA molecules.

The labeling of proteins with fluorescent compounds for microscopy has allowed a greater understanding of biological processes. The preparation of fluorescent proteins is the first step in development of their use in microscopy. Methods are described to label and characterize a protein as an example of the general approach for other proteins. Skeletal muscle alpha-actinin was labeled with either fluorescein-5-maleimide or 5-iodoaceamidofluorescein and the reaction characterized. The maleimide reaction was much more rapid and efficient than the iodoacetamide reaction giving a coupling efficiency of 65% under the given ration conditions. The fluorescein-5-maleimide alpha-actinin was functionally characterized and there was essentially no influence on the fluorescein label on the F-actin binding properties of alpha-actinin. The fluorescein alpha-actinin was also shown to specifically bind to the Z-line of isolated myofibrils. A general outline and discussion are presented on how to label and characterize proteins for use in microscopy.

A freeze-drying device was applied to t-butyl alcohol substituted nerve cells, fibers and synaptic terminals. Ten percent formalin-fixed human cerebellar cortex specimens were postfixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer solution and were rinsed three times in 5% sucrose solution in the same buffer. After the postfixed specimens were dehydrated using a graded series of ethanols and then transferred into a graded series of t-butyl alcohols (freezing point 25.4 degrees C), the t-butyl alcohol substituted specimens were freeze-dried at 15 degrees C and at high vacuum (5 x 10(-2) Torr). The freeze-dried specimens were sputter coated with gold. Scanning electron microscopy revealed synaptic terminals on the surfaces of a Golgi cell and a small flat polygonal cell. Rough somatic surfaces of granule cells were also observed.

In vitro systems and cultured cells are recognized as useful systems in many areas of biomedical research, including X-ray microanalysis. To be reliable, in an vitro system should have an elemental composition close to that of the tissue in situ, react in the same way to stimuli, and retain the in situ regulation of ion transport. In the present paper, four of the most commonly used in vitro systems will be reviewed: incubated tissue slices (liver and pancreas), isolated glands (submandibular gland acini, sweat glands), primary cell cultures (sweat glands, endometrium), and cell lines (the colon cancer cell line T84, immortalized sweat gland cells). Incubation of tissue slices of liver in Krebs-Ringers buffer caused a significant increase in Na and Cl and a decrease in K. Initially, these changes were also observed in the pancreas, but here the values gradually returned to normal. Isolated submandibular gland acini, and isolated sweat gland ducts and coils react in a similar way to stimulation as their in situ counterparts. In primary cultures of coil cells, however, part of the cell population acquires different ion transport characteristics. Technically simplest is the use of cell lines originating from cancer cells (e.g., the T84 cell line) and immortalized cell lines. X-ray microanalysis not only confirms data on ion transport obtained with other techniques, but adds the possibility to investigate the presence of subpopulations within a culture.

We have recently combined immunogold labeling procedures with molecular biology methods to pinpoint the precise locations of nucleic acids in biological material at the ultrastructural level. These new immunocytological approaches involve the incorporation of labeled nucleotides in the nucleic acids present at the surface of ultrathin sections prior to immunogold labeling. The antibodies used recognize a nucleoside analogue (bromodeoxyuridine) or a hapten (biotin) employed to label nucleotides. Examples of high-resolution detection include DNA or RNA present in different substructures of cell nuclei, and in particular, in adenovirus-induced intranuclear regions of HeLa cells. In addition to being highly sensitive and specific, these new methods offer the possibility of studying the spatial distribution of nucleic acids in very well preserved, readily recognizable structures.

The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA.

Whole mount electron microscopy of extracted cells combined with immunogold labeling techniques can be used to characterize the cytoskeletal architecture of cultured cells. As shown with subclones of the C6 rat glioma cell line, heavy metal shadowing was suitable for getting basic information concerning the arrangement of the various filament types within the networks. Pure carbon shadowing combined with immunogold double labeling proved to be optimal to identify linkages between filaments, to localize filament associated proteins and to follow the arrangement of filaments in dense arrays such as lamellipodiae and cell margins. Thin connecting filaments which interact with actin as well as with vimentin filaments and can be labeled with antibodies to the intermediate filament associated protein plectin may play a major role in the structural organization of the cytoskeleton of these cells.

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-associated cytoskeleton ring. By using microtubule altering agents we could demonstrate: (1) the architectural role of microtubules in arranging fenestrae, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the necessity of an intact microtubular cytoskeleton to support the increase in the number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, probably projections of the underlying tubular cytoskeleton.