Zweiter Internationaler Kongreß für Histo- und Cytochemie / Second International Congress of Histo- and Cytochemistry / Deuxième Congrès International d’Histochimie et de Cytochimie pp 165-169 | Cite as
Even though many chromogenic substrates that form color dyes have been found useful with the light microscope, the extension of their use with the electron microscope has had only limited success. The difficulty lies in the more stringent requirements for E.M. localization than those for histochemical purposes. The major requirements are electron scattering ability, low aggregation, and microcrystallinity. This paper reports exploratory chemical studies related to overcome such difficulties. — Polymeric diazonium salts have been synthesized to reduce crystallinity of the azo-dye method. The slow coupling rate and low penetration during incubation have made these substrates unsuccessful for such purpose. The visualization of succinic dehydrogenase activity at E.M. level in sperm cells several years ago (L. Nelson and K. C. Tsou, J. Histochem. Cytochem. 6, 94, 1958) encouraged us to synthesize several polymeric tetrazolium salts for dehydrogenase. Aggregation remains the major problem in extending these substrates for E.M. level for this type of cellular localization. Comparison of the formazans of Nitro-BT, TNBT, and the related polytetrazolium salt at E.M. level provides striking demonstration of the problems involved in such development. Attempts have also been made to demonstrate enzyme sites by the indigo dyes at E.M. level. While chemically hydrolyzed 5-bromo-indoxyl does lead to a more amorphous 5,5'-dibromo indigo, the enzymatically hydrolyzed product tends to crystallize into minute oblong rod crystals which obscure the localization site. However, it was noted with interest that spot electron diffraction patterns on such sites give distinct Laue pattern which can be useful for demonstration of enzyme sites at E.M. level. These preliminary observations provide further impetus in application of histochemical substrates to electron microscopy.
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