SPECTRA AND FLUORESCENCE LIFETIMES OF LISSAMINE RHODAMINE, TETRAMETHYLRHODAMINE ISOTHIOCYANATE, TEXAS RED, AND CYANINE-3.18 FLUOROPHORES - INFLUENCES OF SOME ENVIRONMENTAL-FACTORS RECORDED WITH A CONFOCAL LASER-SCANNING MICROSCOPE

We report on the spectra and fluorescence lifetimes of four commonly u sed fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine is othiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3), Fluorescence lifetime recordings revealed that these spectrally overlapping fluoro phores can be individually detected by their lifetimes, indicating tha t at least four fluorophores can be individually identified in discret e tissue domains by confocal microscopy. A further advantage of lifeti me recordings is that fluorophores that emit light within the same wav elength band can be used and chromatic aberrations are therefore circu mvented, thereby improving the spatial accuracy in imaging of multiple fluorophores, Low and high pH, respectively, tended to influence fluo rophore emission spectra and fluorescence lifetime, IgG conjugation of the fluorophores tended to shift the spectra towards longer wavelengt hs and to change the fluorescence lifetimes. The IgG-conjugated form o f the fluorophores may, when applied to tissue specimens, change the e mission spectrum and lifetime. In addition, different tissue embedding procedures may influence fluorescence lifetime, These observations em phasize the importance of spectral and lifetime characterization of fl uorescent probes within the chemical context in which they will be use d experimentally. Changes in spectra and fluorescence lifetimes may be a useful tool to gain information about the chemical environment of t he fluorophores.