Compact dual-wavelength miniature fluorescence microscope based on spectral multiplexing

Dual-wavelength miniature fluorescence microscopy is desirable for resolving multiple fluorescent signals in freely behaving animal experiments, but existing implementations often require separate detection paths, beam-splitting optics, or additional sensors, thereby increasing system complexity. Here, we report a compact shared-path dual-wavelength miniscope that combines spectral multiplexing with time-division excitation in a single-sensor architecture. The system alternates blue and red excitation in synchrony with camera exposure, encoding fluorescence signals at two wavelengths into an interleaved image stream that is subsequently demultiplexed. The prototype achieves dual-wavelength imaging at an effective frame rate of 30 Hz per wavelength while maintaining a lightweight head-mounted form of approximately 5 g. Imaging experiments using a resolution target, static two-color fluorescent phantoms, and a dynamic flow phantom demonstrate a minimum resolvable line width of 1.74 μm, effective wavelength separation, and dual-wavelength dynamic imaging capability. These results demonstrate that the proposed shared-path, single-sensor design offers a low-complexity approach for compact dual-wavelength miniscope imaging.