Confocal Laser Scanning Microscopic Imaging of Subcellular Organelles, mRNA, Protein Products, and the Microvessel Environment.

Abstract

The confocal laser scanning microscope (CLSM) was employed to visualize highly contrasted images of histochemically stained subcellular organelles. In early studies, only fluorescent signals were detectable by CLSM, however, recent innovations have enabled the visualization of non-fluorescent probes such as horseradish peroxidase (HRP)-diaminobenzidine (DAB). The non-fluorescent signals are permanently preserved and allow repeated or retrospective examinations. We applied CLSM to specimens prepared for light microscopy, and visualized subcellular organelles and pituitary hormone mRNA. The images were comparable to those obtained by electron microscopy (EM). We also applied this technique to the observation of tumor angiogenesis and the microvessel environment of hormone-secreting cells. Both the visualization of subcellular organelles, mRNA and protein products, and 3D images of the microvessel environment of hormone-secreting cells are discussed in this review.

Dynamics of subcellular organelles, growth hormone, Rab3B, SNAP‐25, and syntaxin in rat pituitary cells caused by growth hormone releasing hormone and somatostatin

“…GH immunoreactions developed by DAB were observed under CLSM with the reflection mode, and rab3B immunoreactions developed by ALP-Fuchsin were observed under CLSM with the confocal mode. The methods of observation under CLSM were detailed in our previous reports (Itoh et al, 1997(Itoh et al, , 2001Matsuno et al, 1999Matsuno et al, , 2001a.…”

Histopathological Review of Silent Pituitary Somatotroph Adenoma

“…These paradoxical responses of GH in TRH or GnRH provocation test, which were used as a hallmark of silent somatotroph adenoma in our previous reports [10][11][12][13], are observed primarily in densely granulated somatotroph adenomas. Subcellular organelles and diaminobenzidine signals of immunohistochemical staining can be observed under confocal laser scanning microscopy (CLSM) [2][3][4]. Therefore, when tumor specimens were not available for electron microscopy, CLSM can detect secretory granules using paraffin sections immunostained with anti-GH antibody …”

Simultaneous Detection of DAB and Methyl Green Signals on Apoptotic Nuclei by Confocal Laser Scanning Microscopy

“…The non-fluorescent signals including HRP-DAB-OsO4, osmium black, azo dye, nitroblue tetrazolium (NBT), and heavy metals have advantages over fluorescent signals in that they are permanently preserved and allow repeated or retrospective examination [11,13,14,16]. For example, a 543-nm wavelength helium-neon laser ray illuminated DAB-OsO4 reaction products.…”

“…Recently, the application of CLSM combined with computer image analysis in the field of cell biology has been expanded. CLSM now enables one to observe the subcellular localization of various biologically active substances utilizing routinely processed light microscopic specimens [1-3, 8,13,16,26,33].…”