Scanning microscopy. Supplement最新文献_第2页

Imaging molecular structure of channels and receptors with an atomic force microscope. 用原子力显微镜成像通道和受体的分子结构。

Scanning microscopy. Supplement Pub Date : 1996-01-01

R Lal

{"title":"Imaging molecular structure of channels and receptors with an atomic force microscope.","authors":"R Lal","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"81-95; discussion 95-6"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20521593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Advanced instrumentation and methodology related to cryoultramicrotomy: a review. 冷冻冷冻显微切开术的先进仪器和方法综述。

Scanning microscopy. Supplement Pub Date : 1996-01-01

H Sitte

{"title":"Advanced instrumentation and methodology related to cryoultramicrotomy: a review.","authors":"H Sitte","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"387-463; discussion 463-6"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20521963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Labeling with nanogold and undecagold: techniques and results. 纳米金和非金标记:技术和结果。

Scanning microscopy. Supplement Pub Date : 1996-01-01

J F Hainfeld

引用次数: 0

Simultaneous identification of a specific gene protein product and transcript using combined immunocytochemistry and in situ hybridization with non-radioactive probes. 结合免疫细胞化学和非放射性探针原位杂交，同时鉴定特定基因蛋白产物和转录物。

Scanning microscopy. Supplement Pub Date : 1996-01-01

G V Childs

{"title":"Simultaneous identification of a specific gene protein product and transcript using combined immunocytochemistry and in situ hybridization with non-radioactive probes.","authors":"G V Childs","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"17-24; discussion 24-6"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20521687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation of high efficiency ssDNA hybridization probes by linear polymerase chain reaction (LPCR). 利用线性聚合酶链反应(LPCR)制备高效ssDNA杂交探针。

Scanning microscopy. Supplement Pub Date : 1996-01-01

G W Konat

引用次数: 0

X-ray microscopy: preparations for studies of frozen hydrated specimens. x射线显微镜:冷冻水合标本研究的准备。

Scanning microscopy. Supplement Pub Date : 1996-01-01

A Osanna, C Jacobsen, A Kalinovsky, J Kirz, J Maser, S Wang

{"title":"X-ray microscopy: preparations for studies of frozen hydrated specimens.","authors":"A Osanna, C Jacobsen, A Kalinovsky, J Kirz, J Maser, S Wang","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"349-56; discussion 356-8"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20522053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy. 神经移植用DiI染色和双光子激光扫描显微镜活体成像。

Scanning microscopy. Supplement Pub Date : 1996-01-01

S M Potter, J Pine, S E Fraser

{"title":"Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.","authors":"S M Potter, J Pine, S E Fraser","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"189-99"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2585498/pdf/nihms-72371.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20522143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching. 荧光光谱在细胞成像中的新兴应用:寿命成像、金属配体探针、多光子激发和光猝灭。

Scanning microscopy. Supplement Pub Date : 1996-01-01

J R Lakowicz

{"title":"Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching.","authors":"J R Lakowicz","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"213-24"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20522145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Multiple labeling in electron microscopy: its application in cardiovascular research. 电镜多重标记技术在心血管研究中的应用。

Scanning microscopy. Supplement Pub Date : 1996-01-01

M M Marijianowski, P Teeling, K P Dingemans, A E Becker

{"title":"Multiple labeling in electron microscopy: its application in cardiovascular research.","authors":"M M Marijianowski, P Teeling, K P Dingemans, A E Becker","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"261-71"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20522149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

In situ hybridization, in situ transcription, and in situ polymerase chain reaction. 原位杂交，原位转录和原位聚合酶链反应。

Scanning microscopy. Supplement Pub Date : 1996-01-01

L E De Bault, J Gu

{"title":"In situ hybridization, in situ transcription, and in situ polymerase chain reaction.","authors":"L E De Bault, J Gu","doi":"","DOIUrl":"","url":null,"abstract":"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.","PeriodicalId":77379,"journal":{"name":"Scanning microscopy. Supplement","volume":"10 ","pages":"27-44; discussion 44-7"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20521688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0