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

• Published in: Advanced functional materials 2024-10, Vol.34 (44), p.n/a
• Main Authors: Safaee, Mohammad M., Nishitani, Shoichi, McFarlane, Ian R., Yang, Sarah J., Sun, Ethan, Medina, Sebastiana M., Squire, Henry, Landry, Markita P.
• Format: Article
• Language: English
• Subjects: Biomolecules; Brain; Carbon nanotubes; deep tissue imaging; Femtosecond pulsed lasers; Fluorescence; Heterogeneity; Infrared imaging; Medical imaging; Microscopy; Near infrared radiation; near‐infrared fluorescence microscopy; Photon absorption; Photons; single‐walled carbon nanotubes; Spatial resolution; Thickness; Tissues; two‐photon microscopy; Windows (computer programs)
• ISSN: 1616-301X; 1616-3028
• DOI: 10.1002/adfm.202404709

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, the advantages of NIR and multiphoton imaging are combined by developing a dual‐infrared two‐photon microscope that enables high‐resolution deep imaging in biological tissues. This study first computationally identifies that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. A NIR two‐photon microscope is constructed next with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single‐walled carbon nanotubes (SWNTs). Spatial imaging resolutions are achieved 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. NIR‐II two‐photon microscopy can also measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature this study exploits to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. These results suggest dual‐infrared two‐photon microscopy can 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. A dual‐infrared two‐photon microscope enables high‐resolution deep‐tissue imaging in biological tissues. Utilizing NIR‐II excitation and emission with SWCNTs as fluorophores, this system achieves full‐depth imaging of leaf tissue with minimal autofluorescence and distinguishes Huntington's Disease mouse brain tissues. This approach offers potential for high‐resolution imaging in various challenging biological tissues.