From Optical Fiber Communications to Bioimaging: Wavelength Division Multiplexing Technology for Simplified in vivo Large‐depth NIR‐IIb Fluorescence Confocal Microscopy

Near-infrared II (NIR-II, 900-1880 nm) fluorescence confocal microscopy offers high spatial resolution and extensive in vivo imaging capabilities. However, conventional confocal microscopy requires precise pinhole positioning, posing challenges due to the small size of the pinhole and invisible NIR-II fluorescence. To simplify this, a fiber optical wavelength division multiplexer (WDM) replaces dichroic mirrors and traditional pinholes for excitation and fluorescence beams, allowing NIR-IIb (1500-1700 nm) fluorescence and excitation light to be coupled into the same optical fiber. This streamlined system seamlessly integrates key components-excitation light, detector, and scanning microscopy-via optical fibers. Compared to traditional NIR-II confocal systems, the fiber optical WDM configuration offers simplicity and ease of adjustment. Notably, this simplified system successfully achieves optical sectioning imaging of mouse cerebral blood vessels up to 1000 µm in depth. It can discern tiny blood vessels (diameter: 4.57 µm) at 800 µm depth with a signal-to-background ratio (SBR) of 5.34. Additionally, it clearly visualizes liver vessels, which are typically challenging to image, down to a depth of 300 µm.