Fig. 1: Working principle and linear unmixing of two fluorophores using simulation.
From: Electrochemical fluorescence modulation enables simultaneous multicolour imaging
a, Schematic of the microscope integrated with the electrochemistry setup. The inset shows the input electrochemical potential signal used to modulate the fluorophores between the bright and dim states. A typical three-electrode system is utilized, which includes a transparent ITO-coated glass coverslip as the working electrode (WE), a Ag/AgCl wire in 3 M KCl as the reference electrode (RE) and a platinum mesh as the counter electrode. Each electrode is connected to a potentiostat, which applies a defined potential difference between ITO and RE. b, Fluorescence signals from a STAR RED (blue line) and ATTO 655 (red line) reference dyes, as well as a 1:1 mixture of the two dyes (black line), in response to the potential change. Least-squares fitting with a non-negativity constraint was applied to extract the contribution of each fluorophore from the mixture. The fitting residual is shown below. c, Simulation of the two reference dyes mixed in a 100 × 100-pixel2 image with the indicated patterns. Gaussian noise was added along the vertical axis, ranging from 0% to 50% of the signal intensity. d, The two dyes were successfully unmixed, revealing patterns that correspond to the ground truth. The unmixed images are colour coded and overlaid.
