Short-wavelength excitation two-photon intravital microscopy of endogenous fluorophores

Ting Wu; Jiuling Liao; Feng Xiang; Jia Yu; Yufeng Gao; Lina Liu; Shiwei Ye; Hui Li; Kebin Shi; Wei Zheng

doi:10.1364/BOE.493015

Short-wavelength excitation two-photon intravital microscopy of endogenous fluorophores

Biomed Opt Express. 2023 Jun 14;14(7):3380-3396. doi: 10.1364/BOE.493015. eCollection 2023 Jul 1.

Authors

Ting Wu^{1

2

3}, Jiuling Liao^{1

3}, Feng Xiang^{1

3}, Jia Yu^{1

3}, Yufeng Gao^{1

3}, Lina Liu^{1

3}, Shiwei Ye^{1

3}, Hui Li^{1

3

4}, Kebin Shi^{5

6

7}, Wei Zheng^{1

3

8}

Affiliations

¹ Research Center for Biomedical Optics and Molecular Imaging, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
² University of Chinese Academy of Sciences, No.19(A) Yuquan Road, Shijingshan District, Beijing 100049, China.
³ Shenzhen Key Laboratory for Molecular Imaging, Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology, Shenzhen Institute of Advanced Technology, Shenzhen 518055, China.
⁴ hui.li@siat.ac.cn.
⁵ State Key Laboratory for Mesoscopic Physics and Frontiers Science Center for Nano-optoelectronics, School of Physics, Peking University, Beijing 100871, China.
⁶ National Biomedical Imaging Center, Peking University, Beijing 100871, China.
⁷ kebinshi@pku.edu.cn.
⁸ zhengwei@siat.ac.cn.

Abstract

The noninvasive two-photon excitation autofluorescence imaging of cellular and subcellular structure and dynamics in live tissue could provide critical in vivo information for biomedical studies. However, the two-photon microscopy of short-wavelength endogenous fluorophores, such as tryptophan and hemoglobin, is extremely limited due to the lack of suitable imaging techniques. In this study, we developed a short-wavelength excitation time- and spectrum-resolved two-photon microscopy system. A 520-nm femtosecond fiber laser was used as the excitation source, and a time-correlated single-photon counting module connected with a spectrograph was used to provide time- and spectrum-resolved detection capability. The system was specially designed for measuring ultraviolet and violet-blue fluorescence signals and thus was very suitable for imaging short-wavelength endogenous fluorophores. Using the system, we systematically compared the fluorescence spectra and fluorescence lifetimes of short-wavelength endogenous fluorophores, including the fluorescent molecules tyrosine, tryptophan, serotonin (5-HT), niacin (vitamin B3), pyridoxine (vitamin B6), and NADH and the protein group (keratin, elastin, and hemoglobin). Then, high-resolution three-dimensional (3D) label-free imaging of different biological tissues, including rat esophageal tissue, rat oral cheek tissue, and mouse ear skin, was performed in vivo or ex vivo. Finally, we conducted time-lapse imaging of leukocyte migration in the lipopolysaccharide injection immunization model and a mechanical trauma immunization model. The results indicate that the system can specifically characterize short-wavelength endogenous fluorophores and provide noninvasive label-free 3D visualization of fine structures and dynamics in biological systems. The microscopy system developed here can empower more flexible imaging of endogenous fluorophores and provide a novel method for the 3D monitoring of biological events in their native environment.