Intravital confocal fluorescence lifetime imaging microscopy in the second near-infrared window

We present confocal fluorescence lifetime imaging microscopy in the second near-infrared (NIR-II) window to assess the morphological and biochemical information of live samples. A home-built superconducting single-photon detector (SSPD) was used to facilitate the NIR-II fluorescence lifetime measurement. The SSPD has many advantages, including high sensitivity to NIR-II signals (detection efficiency > 50 % ), fast temporal response ( ∼ 109 p s ), low timing jitter ( ∼ 50 p s ), and low dark count rate ( < 100 c p s ). We demonstrate the feasibility of the developed microscopy system by comparing fluorescence lifetimes of a range of fluorophores with emission in the NIR-II window and by performing multicolor three-dimensional fluorescence lifetime imaging of a mouse ear in vivo . The biochemical properties of the cells and tissues probed by the fluorescence lifetimes of the fluorophores provide complementary information for biomedical studies, significantly benefiting diverse applications in life science.