Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in swiss 3T3 cells

Abstract

This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laserbased confocal microscope, the actual colour of the specimen is viewed directly with the TSM in almost real-time.