Surface reflection interference microscopy: a new method for visualizing cytoskeletal components by light microscopy

Surface reflection interference microscopy of detergent resistant residues of cultured cells stained with protein dyes can be used to obtain high resolution images of the cytoskeleton. We have compared the images obtained using different dyes and have examined the effect of four of these dyes on the visualization of different parts of the cytoskeleton in detail. The dependence of contrast of the images obtained on the illuminating numerical aperture and the wavelength of incident light was determined. Staining with Acid Yellow 36, Guinea Green B and Naphtol Blue Black produces images from the entire cytoskeleton and contrast in these images is relatively insensitive to changes in the incident wavelength. Coomassie Brilliant Blue R250 images, on the other hand, result primarily from reflection from the lower surface of the cytoskeleton and the contrast of these images is sensitive to changes in incident wavelength dropping abruptly in the region of the transmission peak of the stain. From the different spectral sensitivities of the reflection images obtained and from differential interference effects at low and high illuminating numerical apertures, we conclude that the reflection images obtained using the first three stains result from modulation of the reflection by interference effects. In contrast, in the case of Coomassie Brilliant Blue R250 the resulting image originates mainly from selective reflection of wavelength near the absorption range of the dye.