Intravital confocal fluorescence lifetime imaging microscopy in the second near-infrared window
Jia Yu, Rongli Zhang, Yufeng Gao, Zonghai Sheng, Min Gu, Qinchao Sun, Jiuling Liao, Ting Wu, Zhanyi Lin, Peiheng Wu, Lin Kang, Hui Li, Labao Zhang, Wei Zheng
Abstract
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 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mo>></mml:mo> </mml:mrow> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mn>50</mml:mn> </mml:mrow> <mml:mi mathvariant="normal">%</mml:mi> </mml:math> ), fast temporal response ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"> <mml:mo>∼</mml:mo> <mml:mspace width="negativethinmathspace"/> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mn>109</mml:mn> </mml:mrow> <mml:mspace width="thickmathspace"/> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mi mathvariant="normal">p</mml:mi> <mml:mi mathvariant="normal">s</mml:mi> </mml:mrow> </mml:math> ), low timing jitter ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mo>∼</mml:mo> </mml:mrow> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mn>50</mml:mn> </mml:mrow> <mml:mspace width="thickmathspace"/> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mi mathvariant="normal">p</mml:mi> <mml:mi mathvariant="normal">s</mml:mi> </mml:mrow> </mml:math> ), and low dark count rate ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mo><</mml:mo> </mml:mrow> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mn>100</mml:mn> </mml:mrow> <mml:mspace width="thickmathspace"/> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mi mathvariant="normal">c</mml:mi> <mml:mi mathvariant="normal">p</mml:mi> <mml:mi mathvariant="normal">s</mml:mi> </mml:mrow> </mml:math> ). 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.