Laser scanning microscopy in enzyme histochemistry. Visualization of cerium-based and dab-based primary reaction products of phosphatases, oxidases and peroxidases by reflectance and transmission laser scanning microscopy.

The reflectance mode of confocal laser scanning microscopy is suitable to detect cerium-based primary reaction products of oxidases (CeIV-perhydroxide) and phosphatases (CeIII-hydroxy-phosphate converted into CeIV-perhydroxy-phosphate) as well as of DAB-based primary reaction products (Ni-DAB, Ni-FeII-DAB and CeIV-DAB complexes) of cytochrome C oxidase and peroxidases in vibratome, cryotome and semithin plastic sections. In combination with confocal detection 3D images with submicron spatial resolution can be obtained. Moreover, CeIV-perhydroxide, CeIV-perhydroxy-phosphate, CeIV-DAB complexes and catechol-DAB polymers are highly absorptive. Among other additives, especially stable nitroxyl radicals led to a distinct improvement of the DAB staining in terms of sensitivity and proper localization. This was proven in addition by means of blotting a horseradish peroxidase dilution series during several experiments. In sections it was easily possible to record reflectance signals and high transmission contrast at the wavelength of the exciting argon ion laser (preferentially 488 nm). The results of an imbibition study of cerium-containing model precipitates indicate that the cerium generally should be oxidized prior to observation because the index of refraction of CeIV compounds is considerably higher than that of the corresponding CeIII compounds. A comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is possible. Confocal laser scanning microscopy offers a unique way for high resolution detection of primary histochemical reaction products being sufficiently reflective and/or absorptive. The proposed techniques may open new methodological possibilities for basic research and for medical diagnosis.