It is proposed to add excitation fluorescence spectroscopy capability to present microspectrofluorometer with capabilities for topographic and emission fluorescence spectral scans. Thefluorescence excitation attachment will be based on a spinning multizone disk, prepared with a series of alternate light and dark areas such that, as a function of radius, there will be different numbers of alternations at different distances from the center. As the spectrum of the dispersion element is focused on the disk, it will be modulated (coded) according to wavelength (i.e. excitation wavelength interval divided into n 100 nm bands). The resulting coded excitation light falls on the object to be studied. Thus it will be possible to build an excitation-emission matrix (EEMS) of the fluorescence, a powerful method to study complex multi-emission spectra. Both the theory and practice of building such EEMS has been tested, the latter in cultured fibroblasts treated with the fluorescent carcinogenic hydrocarbon benzo(a)pyrene - (BP). It is proposed that the usefulness and applications of the proposed apparatus be investigated in fibroblasts treated with BP metabolized through the cytochrome P450 detoxification system, daunorubicin extruded through the P-glycoprotein-mediated MDR (multidrug-resistance) pathway, or a fluorescent molecule such as mMSA. The observed excitation- emission changes may relate to intracellular metabolization, interactions, binding or quenching of the drug, as well as its elimination by conjugation or extrusion. The microspectrofluorometer complemented to that purpose will also provide real time processing of data enabling alterations of the experimental design in the course of observations. Thus a system of experimentation will be produced which is rapidly responsive to the changes provoked in the cell. NSF FORM 1358 (1/94) 3
