Blind spectral decomposition of single-cell fluorescence by parallel factor analysis

Simultaneous measurement of multiple signaling molecules is essential to investigate their relations and interactions in living cells. Although a wide variety of fluorescent probes are currently available, the number of probes that can be applied simultaneously is often limited by the overlaps among their fluorescence spectra. We developed the experimental system to measure and analyze many overlapping fluorescent components in single cells. It is based on the recording of two-dimensional single-cell fluorescence spectra and on the blind spectral decomposition of fluorescence data by method of parallel factor analysis. Because this method does not require any preknowledge about the shapes of individual component spectra, it can be applied to the specimens that contain fluorescent components with unknown spectra. By examining the performance using the mixture solutions of fluorescent indicators, it was confirmed that >10 largely overlapping spectral components could be easily separated. The effectiveness in the physiological experiments was proven in the applications to the temporal analysis of intracellular Ca2+ concentration and pH, as well as the intrinsic fluorescent components, in single mouse oocytes.