ABSTRACT
Contents Major conceptual breakthroughs have occurred in cellular and molecular biology with the development of light microscopy (LM), scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM), transmission electron microscopy (TEM), and confocal microscopy 0-8493-0815-1/04/$0.00+$ 1.50
(CM). For investigators conducting studies on in situ localization of specific mRNAs or reporter-gene expression in animal or plant tissues and organs, light microscopy and confocal microscopy protocols are required. Scanning electron microscopy allows one to visualize three-dimensional images of the surfaces of whole organisms, organs, tissues, and cells or parts of cells, made possible by the large depth-of-field capabilities of the SEM. X-ray analysis of the distribution of elements present in the cells or tissues can be performed with the SEM as well. The ESEM allows one to observe uncoated, hydrated, living organisms and tissues in the SEM. Subcellular immunolocalization of specific gene products using antibody labels requires knowledge of transmission electron microscopy and specialized techniques of tissue fixation, embedding, and ultrathin sectioning, as well as light microscopy techniques applied via confocal microscopy. In addition to in situ localization, confocal microscopy allows researchers to generate three-dimensional optical sections of living tissues and cells using computer programs for data and section analyses, fluorescent markers, and filter systems that allow investigators to observe fluorescence images of specific proteins, nucleic acids, and ions within whole tissues or organs. Multiphoton microscopy is a new technique that allows the same kind of threedimensional optical sections that one can produce with a confocal microscope, but without the problems of photobleaching, and it allows for deeper penetration due to the longer wavelengths used in this microscope.
