Immunoelectron microscopy and electron microscopic in situ hybridization are undoubtedly the best methods for following the dynamic changes of subcellular organelles; however, these techniques require specific tissue preparation and equipment. More recent developments include a more refined and sophisticated technique, confocal laser scanning microscopy (CLSM), which was originally described by Minsky in 1957 (1) and has since been applied to the field of medical biology. In early experiments, only fluorescent signals were detectable by CLSM (2–9); however, recent innovations have made possible the visualization of nonfluorescent signals such as horseradish peroxidase (HRP) and diaminobenzidine (DAB) signals by CLSM (10,11). Moreover, the combination of CLSM and the image analysis system (IAS) (12) has allowed us to visualize subcellular organelles three-dimensionally in routinely processed light microscopic specimens. We applied CLSM to specimens prepared for light microscopy (12) and demonstrated the intracellular identification of subcellular organelles and pituitary hormone mRNA, comparable to that of electron microscopy (13,14). We also applied CLSM to the study of tumor angiogenesis (15) and the microvessel environment of hormone-secreting cells (16). The visualization of subcellular organelles, mRNA and protein products, as well as three-dimensional images of microvessel environment of hormone-secreting cells is discussed in this chapter.
KeywordsACTH Cell Subcellular Organelle Dichroic Beam Splitter Sodium Chloride Sodium Citrate Biotinylated Oligonucleotide Probe
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