Abstract
We have developed the system to analyze temporal changes of multiple fluorescent components (either intrinsic or extrinsic) in single cells, based on the recording of two-dimensional (excitation x emission wavelengths) spectra. Excitation wavelength was switched using band-pass filters, and emitted fluorescence in a wide range of wavelength was recorded simultaneously with a multi-channel spectrometer. Fluorescence components were extracted from a set of spectral data by the blind spectral decomposition algorithm. In the present study, this system was applied to analyze the fluorescent signals from single mammalian oocytes stained with voltage-sensitive dyes. Several components were separated from time series of 2D spectra recorded from voltage-clamped oocytes whose plasma membrane were stained with a styryl dye, di-8-ANEPPS. Two of them showed a large and opposite voltage dependence, and their ratio could be a good index of membrane potential. The system was further applied to the oocytes in which the endoplasmic reticulum (ER) was stained with another styryl dye, di-18:2-ANEPPS, to investigate the changes in the ER membrane potential during Ca2+ release. The oocytes were loaded with Ca2+-sensitive fluorescent dye, to monitor cytoplasmic Ca2+ simultaneously. The spectral decomposition separated fluorescence components of these dyes successfully, and the result suggested a lumen-negative change in the membrane potential upon IP3-induced Ca2+ release from the ER. [Jpn J Physiol 54 Suppl:S65 (2004)]
