Measuring System Calibration Factors by Unmixing the Excitation–Emission Spectra of One Dish of Cells

Accurate predetermination of the quantum yield ratio (QA/QD) and the extinction coefficient ratio (KA/KD) between acceptor and donor is a prerequisite for quantitative fluorescence resonance energy transfer (FRET) imaging. We here propose a method to measure KA/KD and QA/QD by measuring the excitation–emission spectra (ExEm-spectra) of one dish of cells expressing m (≥3) kinds of FRET constructs. The ExEm-spectra images are unmixed to obtain the weight maps of donor (WD), acceptor (WA), and acceptor sensitization (WS). For each cell, the frequency distribution plots of the WS/WD and WS/WA images are fitted by using a single-Gaussian function to obtain the peak values of WS/WD (SD) and WS/WA (SA). The statistical frequency-SD/SA plots from all cells are fitted by using a multi-Gaussian function to obtain the peak values of both SD and SA, and then the ranges of WS/WD (RSD) and WS/WA (RSA) for each FRET construct are predetermined. Based on the predetermined RSD and RSA values of FRET constructs, our method is capable of automatically classifying cells expressing different FRET constructs. Finally, the WS/WD–WA/WD plot from different kinds of cells is linearly fitted to obtain KA/KD and QA/QD values.