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Backscattered light confocal imaging of intracellular MTT‐formazan crystals
Author(s) -
Bernas Tytus,
Dobrucki Jurek W.
Publication year - 2004
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20070
Subject(s) - formazan , confocal microscopy , mtt assay , confocal , colorimetry , materials science , light scattering , chemistry , microscopy , biophysics , in vitro , scattering , optics , biology , biochemistry , chromatography , physics
Metabolically active animal and plant cells reduce MTT tetrazolium salt to a corresponding nonfluorescent formazan. Reduction of MTT by viable cells is exploited in a number of tests widely used in biological research. The aim of this study was to optimize a microscopy method of detecting small crystals of MTT‐formazan formed in intact cells maintained in in vitro cultures. We examined scattering properties of small intracellular crystals of MTT formazan and found that the efficiency of light scattering was dependent on wavelength. Small (<3 μm) crystals of MTT‐formazan, formed in viable cells, scattered red, but not blue, light. Large crystals, which are formed later at a stage when cells begin to lose viability, scattered both red and blue light. We conclude that optimal detection of early stages of crystallization of MTT‐formazan in living cells is possible using confocal microscopy of red, but not blue, scattered light. High contrast and resolution of images can be achieved by filtering out interference effects in the frequency domain. Microsc. Res. Tech. 64:126–134, 2004. © 2004 Wiley‐Liss, Inc.