This chapter is aimed at those who have not previously done any processing for electron microscopy (EM). It deals with basic preparation of many different types of mammalian material for ultrastructural examination; for processing of plant material (see Hall and Hawes, ref. 1). The material to ...
The production of rapidly frozen thin-film unstained vitrified specimens for cryoelectron microscopy was established as a standard procedure in the early 1980s, primarily because of the persistent efforts of Jacques Dubochet and his colleagues (1-3). Many scientists have now used t ...
Immunocytochemistry is the name given to methods that use antibodies to detect the location of proteins within cells using electron microscopy (EM). The antibodies bind specifically to the protein being investigated and electron opaque markers visualize their location within the c ...
The routine preparation of biological samples for transmission electron microscopy (TEM) usually involves a double-fixation in, first, glutaraldehyde and subsequently in osmium tetroxide (OsO4). The specimens are then dehydrated and embedded in a (heat-polymerized) epoxy re ...
What is high-pressure freezing (HPF)? HPF is a method of specimen preparation for electron microscopy (EM) that freezes noncryoprotected samples up to 0.5 mm in thickness without significant ice crystal damage. Other freezing methods are limited to 1/100 (plunge and impact freezing) to 1/10 ...
It has been demonstrated that enzymes (5,6) can maintain their structure and their activity at very low temperature in concentrated organic solvent. Therefore, in order to minimize molecular thermal vibration, which can have adverse effects on specimens weakly fixed with paraformal ...
The use of microwave energy to reduce processing times during fixation for electron microscopy (EM) is well established in the literature (1-5). The microwave protocol described in this chapter is based on the work of Giberson et al. (6,7) and differs from the microwave literature in that each step ( ...
A new challenge facing electron microscopists in the early 1980s was to combine their experience, taste, and knowledge of the cell ultrastructure with the newly developed antibodies (1) in order to expand morphology into biochemistry and “to bridge the gulf between the so called sciences of m ...
Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distrib ...
Many pure colors and grayscales tones that result from confocal imaging are not reproducible to output devices, such as printing presses, laptop projectors, and laser jet printers. Part of the difficulty in predicting the colors and tones that will reproduce lies in both the computer display, ...
Cell behavior is significantly different in two-dimensional and three-dimensional culture conditions, and a number of methods have been developed to establish and study three-dimensional cellular arrays in vitro. When grown under nonadherent conditions, many types of cells form ...
Three-dimensional (3-D) rendering methods (maximum intensity projection, alpha blending, and isosurface rendering) are described for the visualization of thick, autofluorescent, arthropod cuticular structures (e.g., Drosophila melanogaster external genitalic st ...
The model organism Drosophila melanogaster is particularly well suited for live image analysis. The availability of GFP transgenic flies and a wide array of fluorescent probes, in conjunction with laser scanning confocal microscopy, allow us to image multiple aspects of the cell cycle s ...
The microtubule cytoskeleton plays important roles in a number of cellular processes including cell division, establishing and maintaining cell architecture and polarity, and intracellular trafficking. The identification and characterization of factors required for t ...
The structure of the ureteric tree in developing mouse and rat kidneys has previously been quantified in two dimensions. While this type of analysis may provide evidence of changes in ureteric growth, these measurements are effectively inaccurate, as the ureteric tree is a three-dimensio ...
This article describes a method for quantifying blood flow distribution among lung alveoli. Our method is based on analysis of trapping patterns of small diameter (4 μm) fluorescent latex particles infused into lung capillaries. Trapping patterns are imaged using confocal microsco ...
A confocal microscope was evaluated with a series of tests that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, PMT quality, overall machine stability, and system noi ...
The first practical laser scanning confocal microscopes were introduced to the biomedical community over 30 years ago. Their subsequent development continues to influence the introduction of new methods and applications of optical sectioning microscopy.
In a few short years, the Internet (in terms of the World Wide Web) has become a powerful informational resource for the original scientific literature pertaining to biological investigations using the laser scanning confocal microscope. However, there still remains an obvious void in the ...
The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluoresce ...
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