Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared

Significance Shortwave infrared (SWIR) fluorescence imaging is a tool for visualizing biological processes deep within tissue or living animals. Our study shows that the contrast in a SWIR fluorescence image is primarily mediated by the absorptivity of the tissue, and can therefore be tuned through deliberate selection of imaging wavelength. We show, for example, that, in 3D tissue phantoms and in brain vasculature in vivo in mice, imaging at SWIR wavelengths of the highest water absorptivity results in the greatest fluorescence contrast. We further demonstrate, in microscopy of ex vivo mouse liver tissue, that imaging at wavelengths of high tissue absorptivity can also increase imaging penetration depth, and use a theoretical contrast model to explain this effect. Recent technology developments have expanded the wavelength window for biological fluorescence imaging into the shortwave infrared. We show here a mechanistic understanding of how drastic changes in fluorescence imaging contrast can arise from slight changes of imaging wavelength in the shortwave infrared. We demonstrate, in 3D tissue phantoms and in vivo in mice, that light absorption by water within biological tissue increases image contrast due to attenuation of background and highly scattered light. Wavelengths of strong tissue absorption have conventionally been avoided in fluorescence imaging to maximize photon penetration depth and photon collection, yet we demonstrate that imaging at the peak absorbance of water (near 1,450 nm) results in the highest image contrast in the shortwave infrared. Furthermore, we show, through microscopy of highly labeled ex vivo biological tissue, that the contrast improvement from water absorption enables resolution of deeper structures, resulting in a higher imaging penetration depth. We then illustrate these findings in a theoretical model. Our results suggest that the wavelength-dependent absorptivity of water is the dominant optical property contributing to image contrast, and is therefore crucial for determining the optimal imaging window in the infrared.