Scanning microscopy. Supplement最新文献

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding, its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces.

The purpose of this paper is to describe the key variables in sample and reagent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixative, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near confluence, then physically removed and processed. When possible three samples (4 microM tissue sections or 1-5000 cells) should be placed on silane coated glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DNA detection by PCR in situ hybridization whereas optimal protease digestion time is variable and related to formalin fixation time for reverse transcriptase (RT) in situ PCR. RT in situ PCR requires an overnight digestion with DNase. The amplifying solution should contain 4.5 mM MgCl2, 0.05% bovine serum albumin, and, for RNA analysis, the reporter nucleotide. A false positive signal would be evident with incorporation of the reporter nucleotide for DNA targets due to DNA repair; this can be avoided with frozen, fixed tissues and the hot start maneuver. Otherwise, one needs to use a labeled probe and a hybridization step to detect amplified DNA targets in paraffin embedded tissues.

The introduction of video rate confocal laser scanning microscopes (VRCLSM) used in reflection mode with high magnification, high aperture objective lenses and with further magnification by a zoom facility allowed the first detailed observations of the activity of living cytoplasm and offered a new tool for investigation of the structural transition from the living state to the specimen fixed for electron microscopy (EM). We used a Noran Odyssey VRCLSM in reflection (backscattered) mode. A greater degree of oversampling and more comfortable viewing of the liver or taped video image was achieved at zoom factor 5, giving a display monitor field width of 10 microns. A series of mesenchyme derived cell lines--from normal cells to sarcoma cells of different malignancy--was used to compare behaviour of the observed intracellular structures and results of fixation. We contrasted the dynamic behaviour of fine features in the cytoplasm of normal and neoplastic living cells and changes induced by various treatments. The tubulomembraneous 3D structure of cytoplasm in living cells is dynamic with motion observable at the new limits of resolution provided by VRCLSM. All organelles appear integrated into one functional compartment supporting the continuous 3D trafficking of small particles (vesicles). This integrated dynamic spatial network (IDSN) was found to be largest in neoplastic cells.

Confocal laser scanning microscopy (CLSM) and intermediate voltage transmission electron microscopy (IVEM) each has its own particular advantages. CLSM can examine living cells, but is particularly useful when applied to cells that have been lightly fixed, permeabilized, and stained with fluorescent-labeled antibodies for localization of specific molecular species at the resolution of the light microscope while still in the hydrated state. IVEM provides much higher resolution images, but requires more drastic preparation procedures, including dehydration. This paper presents methods for combining these complementary approaches to examine exactly the same cells sequentially by CLSM and IVEM. Cells are grown in culture on sterile formvar films spread over gold index grids on cover glasses, which are mounted on larger cover glasses or microscope slides with spacers to prevent compression of the cells. Light and epifluorescence microscopy, and CLSM are performed concentrating on cells in grid openings. Then the grids are fixed with aldehydes followed by OsO4, dehydrated and critical point dried (CPD) from liquid CO2. Immediately following CPD, the grids are ready for examination in the IVEM. Low magnification (300-600x) survey images allow correlation of the IVEM images with the light microscopic images. In higher power images, structures that are fluorescent labeled can be related to corresponding regions in the IVEM images.

Although much information about chromosome structure and behaviour has been obtained using light microscopy, greater resolution is needed for a thorough understanding of chromosome organisation. Scanning electron microscopy (SEM) can provide valuable data about these three-dimensional organelles. The introduction of methods using osmium impregnation of methanol-acetic acid-fixed chromosome spreads revolutionised matters, producing life-like images of chromosomes. Nevertheless, it became clear that osmium impregnation introduced various artefacts, although the resulting images were still useful. Methanol-acetic acid-fixed chromosomes are, in fact, flattened on the glass substratum, and the 3-dimensional appearance obtained after osmium impregnation is the result of swelling during this process. At the same time, the fibrous substructure of the chromosomes becomes much coarser. More recently a number of alternative methods have become available for studying chromosomes by SEM. Isolated chromosomes, that have not been allowed to dry during preparation, retain a 3-dimensional appearance without osmium impregnation, and the same is true of methanol-acetic acid-fixed chromosomes that have been treated with 45% acetic acid and processed without drying; however, these methods do not permit the routine production of intact metaphase spreads. Use of cytocentrifuge preparations obviates the use of acetic acid fixation and osmium impregnation, produces intact metaphase spreads, and permits the immunocytochemical detection of antigens that are easily destroyed by routine fixation procedures.

Two new simple stabilization procedures for freeze-dried biological material are introduced which are compatible with low temperature embedding (LTE) in Lowicryl. The first method uses a Lowicryl K11M/HM20 mixture supplemented with 0.3% uranyl acetate for LTE. For the second method polymerized Lowicryl blocks containing the freeze-dried material are exposed to OsO4 vapor which penetrates into the Lowicryl block and stabilizes the embedded specimen. The quality of structural preservation is demonstrated with human leukocytes.

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA supercoiling were analyzed using electron microscopy. In parallel, this Mg2+/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or in liquid by scanning force microscopy. An advantage of such a correlative approach is that parallel imaging can reveal complementary information. The benefit of such a combined approach in analysis of protein-induced DNA bending is discussed.

By modifying freeze-fracture replication, a standard electron microscopy fixation technique, for use with the scanning tunneling microscope (STM), a variety of soft, non-conductive biomaterials can be imaged at high resolution in three dimensions. Metal replicas make near ideal samples for STM in comparison to the original biological materials. Modifications include a 0.1 micron backing layer of silver and mounting the replicas on a fine-mesh silver filters to enhance the rigidity of the metal replica. This is required unless STM imaging is carried out in vacuum; otherwise, a liquid film of contamination physically connects the STM tip with the sample. This mechanical coupling leads to exaggerated height measurements; the enhanced rigidity of the thicker replica eliminates much of the height amplification. Further improvement was obtained by imaging in a dry nitrogen atmosphere. Calibration and reproducibility were tested with replicas of well characterized bilayers of cadmium arachidate on mica that provide regular 5.5 nm steps. We have used the STM/replica technique to examine the ripple shape and amplitude in the P beta phase of dimyristoylphosphatidyl-choline (DMPC) in water. STM images were analyzed using a cross-correlation averaging program to eliminate the effects of noise and the finite size and shapes of the metal grains that make up the replica. The correlation averaging allowed us to develop a composite ripple profile averaged over hundreds of individual ripples and different samples. The STM/replica technique is sufficiently general that it can be used to examine a variety of hydrated lipid and protein samples at a lateral resolution of about 1 nm and a vertical resolution of about 0.3 nm.

The recently discovered high lateral conductivity of molecularly thin adsorbed water films enables investigation of biological specimens, and even of surfaces of hydrophilic insulators by scanning tunneling microscopy (STM). Here we demonstrate the capabilities of this method, which we call hydration-STM (HSTM), with images of various specimens taken in humid atmosphere: We obtained images of a glass coverslip, collagen molecules, tobacco mosaic virus, lipid bilayers and cryosectioned bovine achilles tendon on mica. To elucidate the physical mechanism of this conduction phenomenon we recorded current-voltage curves on hydrated mica. This revealed a basically ohmic behavior of the I-V curves without a threshold voltage to activate the current transport and indicates that electrochemistry probably does not dominate the surface conductivity. We assume that the conduction mechanism is due to structuring of water at the surface.