Multicolor imaging based on brightness coded set

Fluorescence imaging necessitates precise matching of excitation source, dichroic mirror, emission filter, detector and dyes, which is complex and time-consuming, especially for applications of probe multiplexing. We propose a novel method for multicolor imaging based on a brightness coded set. Each brightness code consists of 12 bits ($OOOXXXYYYZTT$), denoting probe type, cube, emission filter, imaging result and priority, respectively. The brightness of a probe in an imaging system is defined as the product of extinction coefficient, quantum yield and the filter transmittance. When the brightness exceeds the threshold, $Z=1$ indicates a clear image, otherwise $Z=0$. The higher the brightness value the higher the priority (TT). To validate the efficacy and efficiency of the coding method, we conducted two separate experiments involving four-color imaging. The proposed method offers a substantial simplification of the conventional approach to device matching in multicolor imaging by leveraging spectrograms, and presents a promising avenue for the advancement of intelligent multicolor imaging systems.