实验仪器使用技术

Regulation of cell volume is a fundamental homeostatic mechanism in the face of osmotic stress (1,2). One approach to understanding aspects of cell volume regulation involves the removal of cells from the matrix and manipulation in culture (3). Measurements are often also needed from cells w ...

Confocal laser scanning microscopy (CLSM) can be used to obtain optical sections of thick tissues that are relatively free of interfering autofluorescence, and that do not strongly scatter or absorb either the excitation or emission light. This chapter provides protocols used to exami ...

The major application of confocal microscopy in the biomedical sciences is for imaging either fixed or living tissues that have usually been labeled with one or more fluorescent probes. When these samples are imaged using a conventional light microscope, the fluorescence in the specimen a ...

Accurate alignment of the microscope is vital for most measurements. Details of the adjustments will vary from system to system but the principles of alignment are universal. The methods given here should be appropriate for any spot-scanning confocal microscope but will not be directly ap ...

Confocal microscopy is routinely used to produce high-resolution images of single, double-, and triple labeled fluorescent samples. The images are collected as single optical sections (2D imaging), as Z-series (3D imaging), as time-lapse series (2D over time), or as Z-series over time (3D over ...

In this chapter we describe a simple method to prepare 3D illustrations from stereo-pairs of images from confocal sections. Methods for preparing both stereo slides and stereo images for publication using Microsoft’s PowerPoint� 97 and CorelDraw� 8 are described. The parallel and cross- ...

Imaging methods, whether they use film, videotape, or digital capture methods, are only of use if you are able to readily categorize and retrieve the information and images that are produced. Modern imaging techniques are capable of generating large numbers of images of various dimensions and ...

Visualizing change over time is essential to understanding biological events such as embryogenesis and development. More and more specimens can be visualized in the living state due to improvements in confocal microscopy, and the development of novel probes such as the green fluoresce ...

Conventional microscopy delivers two-dimensional images in real time and real color to the eye of the user. Confocal microscopy adds a third dimension by imaging only one plane within the sample at a time so that variations in depth can be quantified (1). This has both positive and negative aspects. T ...

The merger of multiple immunoflurescent labeling and the laser scanning confocal microscope (LSCM) has greatly enhanced experimentation in many areas of biomedical research. Recently, genetic tools have become available for marking individual cells, or cells within a tissue of ge ...

This chapter differs from others in this volume in that it does not describe protocols per se. Rather, it consists of checklists, cautions, tips, rules of thumb, and advice related to fluorescent probes in general and probes for confocal microscopy in particular.

In situ hybridization within whole-mount Drosophila tissues was made routine with the introduction of digoxigenin labeled probes and alkaline phosphatase based detection methods (1). However, this method of detection until recently has been limited by the required use of alkaline p ...

Confocal scanning microscopy has been successfully used for immunofluorescence work in yeast. The major axis of a Saccharomyces cerevisiae haploid cell is approx 4 μm. Optical sections of approximately 2� 0.4 μm thickness from fluorescently-labeled yeast cells can be obtained using t ...

The increasing availability of confocal microscopy has begun a revolution in plant biology in which microscopy has again become a powerful tool for understanding structure and function. Examples of applications include: three-dimensional (3D) reconstruction of the interphase ...

The use of antibodies to visualize the distribution and subcellular localization of gene products powerfully complements genetic and molecular analysis of gene function in Caenorhabditis elegans. Double and triple staining protocols are particularly useful for several rea ...

Imaging animal fertilization presents a number of unique challenges that arise from the unusual nature of the cells involved. Eggs are among the largest cells produced by animals, and sperm are frequently the smallest. Observation of the initial steps of fertilization, including sperm- ...

One can easily distinguish between two general modes of operation of the atomic force microscope (AFM) depending on absence or presence in the instrumentation of an additional device that forces the cantilever to oscillate in the proximity of its resonant frequency. The first case is usually ...

Microscopes have always been one of the essential instruments for research in the biomedical field. Radiation-based microscopes (such as the light microscope and the electron microscope) have become trustworthy companions in the laboratory and have contributed greatly to our sci ...

Images taken with the atomic force microscope (AFM) originate in physical interactions that are totally different from those used for image formation in conventional light and electron microscopy. One of the effects is that a new series of artifacts can appear in images that may not be readily re ...

A large body of recent literature describes the use of atomic force microscopy (AFM; ref. 1) for the study of living cells. These experimental findings clearly indicate that AFM is a very valuable tool for the 3D imaging of flat biological samples strongly adhering to a substrate, with a lateral resol ...