Selecting an optimal imaging window for NIR fluorescence wide-field microscopy

Near-infrared (NIR) fluorescence imaging has become indispensable in biomedical research owing to its deep penetration and high contrast. Selecting an appropriate imaging window is crucial to maximizing the positive effect of light absorption for achieving the highest signal-to-background ratio. Existing studies on imaging window selection mainly focus on low numerical aperture (NA) macroscopic systems; however, scattered light plays a decisive role in high-NA microscopic systems, thereby leaving a critical gap in current understanding. To address this, we propose the concept of best-matched absorption as a universal criterion for window optimization across diverse imaging conditions in NIR fluorescence wide-field microscopy. By combining Monte Carlo simulations with a custom-built microscope, we systematically quantify how this best-matched absorption coefficient varies with system NA, imaging depth, and probe brightness. Our results reveal that higher NA or elevated probe brightness induces a redshift in the optimal imaging window, which is generalized for both ideal probes and the clinically used indocyanine green. Moreover, we develop tailored window selection strategies for challenging imaging conditions including superficial occlusions, deep background, and targets extending beyond the depth of field. This study establishes a versatile framework for optimizing image quality in NIR fluorescence wide-field microscopy, with direct significance in biomedical applications.