Fig. 1: Schematic of 3D-SpecDIM.

a The focused laser spot is rapidly scanned within a small volume after the objective lens. The molecule’s deviation from the center of the illumination volume is estimated in real time using an FPGA, which processes photon arrival time information. Based on this deviation, a feedback control voltage is applied to the piezo stage to reposition the molecule at the center of the illumination volume. This process continuously locks the target molecule within the small excitation volume, enabling high-spatiotemporal-precision 3D single-molecule dynamics observation. b To simultaneously acquire the single-molecule fluorescence spectrum, a prism-based spectral imaging system was integrated into the detection path. The fluorescence was dispersed by a prism and projected onto an EMCCD to capture the spectral profile. The target-locking imaging strategy enables spectral acquisition with high imaging speed, high spectral precision, and large imaging depth. c The synchronization of 3D positional dynamics and spectral dynamics allows for multiparameter dynamic data acquisition.