Scan-less full-field fluorescence-lifetime dual-comb microscopy using two-dimensional spectral mapping and frequency multiplexing of dual-optical-comb beats

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for quantitative fluorescence imaging because fluorescence lifetime is independent of concentration of fluorescent molecules or excitation/detection efficiency and is robust to photobleaching. However, since FLIM is based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. In this article, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed RF signals. A vast number of dual-optical-comb beats between dual optical frequency combs is effectively adopted for 2D spectral mapping and high-density frequency multiplexing in radio-frequency region. Bimodal images of fluorescence amplitude and lifetime are obtained with high quantitativeness from amplitude and phase spectra of fluorescence RF comb modes without the need for mechanical scanning. The proposed method will be useful for rapid quantitative fluorescence imaging in life science.