Long‐term observation of cultured cells by interference‐reflection microscopy: Near‐infrared illumination and Y‐contrast image processing

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Abstract
Interference‐reflection microscopy (IRM) is the only method presently available with which to visualize cell‐substratum adhesions in living tissue culture cells continuously for long periods of time without the use of fluorescent markers (Curtis: J. Cell Biol. 20:199–215, 1964; Izzard and Lochner: J. Cell Sci. 21:129–159, 1976). This method utilizes approximately 1% of the incident illumination to produce the IRM image (Verschueren: J. Cell Sci. 75:279–301, 1985) and so far has required the use of high‐intensity light sources in the visible spectral range (400–800 nm). Unfortunately, visible light of this intensity and spectral range induces marked changes in the behavior and morphology of motile fibroblasts, including cessation of locomotion. In contrast, the present paper reports that continuous observations of live cells in IRM for periods of up to 8 hours are possible if the illuminating light is in the red to near‐infrared range (650–950 nm) and without any observable change in normal cell morphology or behavior. In addition, we describe how the technique of Y‐contrast image processing can be applied to IRM images to create a three‐dimensional image of the ventral cell surface topography.