Scanning microscopy. Supplement最新文献

Biological membranes contain specialized protein macromolecules such as channels, pumps and receptors. Physiologically, membranes and their constituent macromolecules are the interface surfaces toward which most of the regulatory biochemical and other signals are directed. Yet very little is known about these surfaces. The structure of biological membranes has been analyzed primarily using imaging techniques that are limited in their resolution of surface topology. An atomic force microscope (AFM) developed by Binnig, Quate and Gerber, can image molecular structures on specimen surfaces with subnanometer resolution, under diverse environmental conditions. Also, AFM can manipulate surfaces with molecular precision: it can nanodissect, translocate, and reorganize molecules on surface. The surface topology has been imaged for several hydrated channels, pumps and receptors which were a) present in isolated native membranes, b) reconstituted in artificial membrane or, c) expressed in an appropriate expression system. These images, at molecular resolution, reveal exciting new findings about their architecture. AFM induced "force dissection" reveals surfaces which are commonly inaccessible. In whole cell studies, in addition to the molecular structure of membrane receptors and channels, correlative electrical and biochemical activities have been examined. Such study suggests a "single cell" experiment where the structure-function correlation of many cloned channels and receptors can be understood.

This review is concerned with the considerable progress in the field of cryo-ultramicrotomy (cryofixation, cryosectioning, investigation and analysis of cryosections) during recent years. This progress includes both more efficient instrumentation and methodology. The article is mainly directed to the investigation and analysis of frozen-hydrated sections in the low dose cryo-transmission electron microscopy (TEM) and cryo-energy filtered TEM (EFTEM). A general survey is followed by an evaluation of the different relevant procedures. Both cryo-ultramicrotomy for macromolecular cytochemistry (Tokuyasu technique) and cryo-ultramicrotomy for element analysis are only shortly mentioned without discussion of the chemical and analytical approach. Because of lack of first hand experience, cryo-sectioning for X-ray microanalysis in the frozen-hydrated state according to Hall and Gupta is not included into this review. The methods and instruments required for ultrathin sectioning at low temperatures are described and discussed in detail. This concerns the preceding cryofixation, the cryosectioning itself with special emphasis to the required stability and precision of the cryo-ultramicrotome, the characteristics of the knives, the charging phenomena due to sectioning and the subsequent TEM investigation including EFTEM with electron spectroscopic imaging (ESI) and the available accessories for digital low dose registration of signals.

A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and allow many new probes to be made with peptides, nucleic acids, lipids, drugs, and other molecules. A new fluorescent-gold conjugate is useful for examining localization by fluorescence microscopy, then visualizing the same label at the ultrastructural level in the electron microscope.

Simultaneous identification of messenger RNA (mRNA) and proteins in the same cells or tissues is a valuable tool to help the cell biologist evaluate the cell secretory cycle. Some cells may produce the mRNA and delay the production of the proteins. Alternatively, the proteins may be rapidly secreted. Other cells may produce both in sequence within the same time frame. Because of this difference, some cells can only be identified by their mRNA product. Others may have both products. This presentation describes a non-radioactive approach to the detection of both products with dual-peroxidase labeling protocols in use in this laboratory since 1983. The first detection system uses biotinylated cRNA probes or oligoprobes in in situ hybridization along with antisera to biotin to detect the hybrid. The detection system is amplified by 2-3 layers of anti-biotin, second antibody (made against the anti-biotin) and streptavidin conjugated to horseradish peroxidase. After the mRNA is detected with a blue-black substrate (nickel intensified diaminobenzidine), the antigens are detected with immunoperoxidase techniques and orange-amber substrate. The in situ hybridization protocol can also be used at the electron microscopic level. Trouble shooting and control protocols are also described. This approach has been shown to be valuable for detection of pituitary hormones, growth factors mRNAs and antigens.

The polymerase chain reaction (PCR) methodology can be employed to produce DNA hybridization probes. The major advantages of this paradigm over other techniques include superior specific activity of the probes, the versatility of sequence selection, the ability to produce short probes, and the simplicity of the procedure. We have further improved the efficiency of PCR probes by generating single stranded (ssDNA) probes that do not reanneal with themselves in solution, and hence, their availability for the interaction with the complementary sequences of the target is profoundly increased. Protocols for 32P-dCTP labeled and digoxigenin-dUTP labeled probes have been elaborated to maximize the incorporation rate of the label as well as to provide for the production of full-length probes. The ssDNA probes may be particularly suitable for nucleic acid detection in tissues by in situ hybridization.

X-ray microscopes provide higher resolution than visible light microscopes. Wet, biological materials with a water thickness of up to about 10 microns can be imaged with good contrast using soft X-rays with wavelengths between the oxygen and carbon absorption edges (at 24 and 43 A). The Stony Brook group has developed and operates a scanning transmission X-ray microscope (STXM) at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The microscope is used for imaging with a current resolution of 50 nm, and for elemental and chemical state mapping. Radiation damage imposes a significant limitation upon high resolution X-ray microscopy of room temperature wet specimens. Experience from electron microscopy suggests that cryo techniques allow vitrified specimens to be imaged repeatedly. This is due to the increased radiation stability of biological specimens in the frozen hydrated state. Better radiation stability has been shown recently with a cryo transmission X-ray microscope developed by the University of Göttingen, operating at the BESSY storage ring in Berlin, Germany. At Stony Brook, we are developing a cryo scanning transmission X-ray microscope (CryoSTXM) to carry out imaging and spectro-microscopy experiments on frozen hydrated specimens. This article will give an outlook onto the research projects that we plan to perform using the CryoSTXM.

We are developing a multielectrode silicon "neuroprobe" for maintaining a long-term, specific, two-way electrical interface with nervous tissue. Our approach involves trapping a neuron (from an embryonic rat hippocampus) in a small well with a stimulation/recording electrode at its base. The well is covered with a grillwork through which the neuron's processes are allowed to grow, making synaptic contact with the host tissue, in our case a cultured slice from a rat hippocampus. Each neuroprobe can accommodate 15 neurons, one per well. As a first step in studying neurite outgrowth from the neuroprobe, it was necessary to develop new staining techniques so that neurites from the probe neurons can be distinguished from those belonging to the host, without interference from non-specific background staining. We virtually eliminated background staining through a number of innovations involving dye solubility, cell washing, and debris removal. We also reduced photobleaching and phototoxicity, and enhanced imaging depth by using a 2-photon laser-scanning microscope. We focused on using the popular membrane dye, DiI, however a number of other membrane dyes were shown to provide clear images of neural processes using pulsed illumination at 900 nm. These techniques will be useful to others wishing to follow over time the growth of neurons in culture of after transplantation in vivo, in a non-destructive way.

Advances in time-resolved fluorescence spectroscopy can be applied to cellular imaging. Fluorescence lifetime imaging microscopy (FLIM) creates image contrast based on the decay time of sensing probes at each point in a two-dimensional image. FLIM allows imaging of Ca2+ and other ions without the need for wavelength-ratiometric probes. Ca2+ imaging can be performed by FLIM with visible wavelength excitation. Instrumentation for FLIM is potentially simple enough to be present in most research laboratories. Applications of fluorescence are often limited by the lack of suitable fluorophores. New, highly photostable probes allow off-gating of the prompt autofluorescence, and measurement of rotational motion of large macromolecules. These luminescent metal-ligand complexes will become widely utilized. Modern pulse lasers allow new experiments based on non-linear phenomena. With picosecond and femtosecond lasers fluorophores can be excited by simultaneous absorption of two or three photons. Hence, Ca2+ probes, membrane probes, and even intrinsic protein fluorescence can be excited with red or near infrared wavelengths, without ultraviolet lasers or optics. Finally, light itself can be used to control the excited state population. By using light pulses whose wavelength overlaps the emission spectrum of a fluorophore one can modify the excited state population and orientation. This use of non-absorbed light to modify emission can have wide reaching applications in cellular imaging.

The heart is a muscular pump kept together by a network of extracellular matrix components. An increase in collagens, as in chronic congestive heart failure (CHF), is thought to have a negative effect on cardiac compliance and, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electron microscopy (SEM) can put these structures in three-dimensional perspective. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this technique in our studies on heart failure. Heart specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated by the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjugated secondary antibodies or protein-A gold conjugates. Depending on the objective, ultra small gold (USG) conjugates or a regular probe size can be used. Labeling efficiency could be increased by bridging antibodies. The double and triple staining procedures were based on single staining methods using one- and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labeling, allowed a detailed study of the organization of the extracellular matrix and its relationship with cardiac myocytes. This may prove to be a useful tool for the study of chronic heart failure.

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR (ISPCR), it is the repeated in situ duplication of both the sense and antisense strands of DNA to increase the number of copies of the target sequence. ISH, IST, and ISPCR each have their advantages and disadvantages. The purpose of this chapter is to address in situ considerations required of these techniques, emphasizing tissue fixation, pre-hybridization steps, DNA probes, RNA probes, oligoprobes, and probe labeling. Five successfully used protocols are presented as examples. Any given nucleotide target sequence may have its own unique set of optimum conditions, thus requiring some adjustment in the hands of the user.