Observation of near-infrared fluorescence from within biological specimens using X-ray light-sheet microscopy for deep-tissue imaging

Abstract Light-sheet fluorescence microscopy enables rapid three-dimensional imaging of biological specimens with low phototoxicity and high signal-to-noise ratio, but its imaging depth is limited by optical scattering and absorption in tissue. X-ray light-sheet microscopy, termed Microscopy by Achromatic X-rays With Emission of Laminar Light (MAXWELL), addresses this limitation by using X-rays as a deeply penetrating excitation source. In this study, we extended MAXWELL to near-infrared (NIR) fluorescence detection, taking advantage of the reduced tissue scattering of NIR light compared with visible wavelengths. Screening of Er³⁺/Yb³⁺-codoped rare-earth ceramic phosphors identified Y₂O₃:Yb, Er as the composition producing the highest camera-detected NIR signal. In uncleared mouse brain vasculature, the analysis yielded observable depths of 300 μm for visible detection and 800 μm for NIR detection, corresponding to approximately 2.7-fold greater depth. Phantom measurements showed less lateral signal broadening in the NIR channel than in the visible channel, while the NIR signal attenuated more steeply with depth. Comparison of the phosphor emission and water absorption spectra suggested that this depth dependence reflects a combination of scattering and wavelength-dependent absorption. These proof-of-concept results establish the feasibility of NIR detection in MAXWELL and identify detector response, phosphor brightness, and tissue optical attenuation as joint determinants of deep-tissue performance.