Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source

Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely complicated or slow for in vivo imaging. Herein, we propose a novel high-speed multimodal optical microscope system that simultaneously visualizes five different microscopic contrasts, i.e., two-photon excitation, second-harmonic generation, backscattered light, near-infrared fluorescence, and fluorescence lifetime, using a single femtosecond pulsed laser. Our proposed system can visualize five modal images with a frame rate of 3.7 fps in real-time, thereby providing complementary optical information that enhances both structural and functional contrasts. This highly photon-efficient multimodal microscope system enables various properties of biological tissues to be assessed.