A new UV–visible confocal laser scanning microspectrofluorometer designed for spectral cellular imaging

With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 × 0.5 × 1 μm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342). © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 101–115, 1999