174 / 2024-06-14 17:03:30

Short-wavelength excitation two-photon microscopy of endogenous fluorophores

two-photon microscopy,autofluorescence,fluorescence imaging

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, high-resolution three-dimensional (3D) label-free imaging of different biological tissues was performed in vivo or ex vivo. Furthermore, 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.