Green fluorescent protein (GFP) and its variants are highly convenient markers to trace the movement of proteins in living cells. Their fluorescence, however, is not strong enough at physiological temperatures for mammalian cells, and the blue variant, blue fluorescent protein (BFP), is photo-bleached quickly. To overcome these limitations, we introduced random mutations to the BFP sequence by mutagenic PCR, and selected clones that fluoresce brighter, after prolonged exposure to UV light. One of such clones had an amino acid substitution adjacent to the chromophore. By combining this mutation with mutations known to improve the fluorescence intensity, we obtained GFP and BFP variants having remarkably stronger fluorescence when expressed in mammalian cells as well as in Eschelichia coli. These mutants have increased stability and are present in the active species in higher proportions at 37°C than are the original proteins. Thus, they would be highly convenient tools for mammalian cell biology.

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We have improved the optics of the differential interference contrast (DIC) microscope for the purpose of using infrared illumination in order to get better resolution and clearer images of cells in living preparations than those obtained with the conventional IR-DIC microscope without improvements. Two main improvements were introduced to pass the infrared illumination through the critical optical components of the original DIC microscope, so that DIC performance has been successfully achieved within the wavelength range of infrared illumination. Application of the present IR-DIC video microscopy to the observation of cells in the living retina and the cerebellar slice preparation of Wistar rats allowed the viewing of cells below the surface of the preparations at a depth l.5 times greater than previously observed for IR-DIC video microscopy in the past, with better resolution and clearer images. In particular, high-quality images of cell layers of the living retina were obtained and suggest interesting possibilities for future research in this field. The technical details of the improved lR-DIC microscope and the results of the observations obtained by using the present microscope are reported here, together with a discussion of a variety of applications to biological and biophysical research.

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