Dual Infrared 2-Photon Microscopy Achieves Minimal Background Deep Tissue Imaging in Brain and Plant Tissues.

Traditional deep fluorescence imaging has primarily focused on red-shifting imaging wavelengths into the near-infrared (NIR) windows or implementation of multi-photon excitation approaches. Here, we combine the advantages of NIR and multiphoton imaging by developing a dual-infrared two-photon microscope to enable high-resolution deep imaging in biological tissues. We first computationally identify that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. We next construct a NIR two-photon microscope with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single-walled carbon nanotubes (SWNTs). We achieve spatial imaging resolutions close to the Abbe resolution limit and eliminate blur and background autofluorescence of biomolecules, 300 μm deep into brain slices and through the full 120 μm thickness of a Nicotiana benthamiana leaf. We also demonstrate that NIR-II two-photon microscopy can measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature we exploit to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. Our results suggest dual-infrared two-photon microscopy could accomplish in-tissue structural imaging and biochemical sensing with a minimal background, and with high spatial resolution, in optically opaque or highly autofluorescent biological tissues.