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. •The image quality is characterized by the actual brightness value of the probe in a specified microscopic imaging system.•Coding of imaging devices, probes, and brightness for quantitative and rapid screening of imageable probes and their configurations.•The brightness coded sets enable rapid screening for multicolor imaging, advancing intelligent multi-color fluorescence microscopy systems.