In vivo label-free two-photon excitation autofluorescence microscopy of microvasculature using a 520 nm femtosecond fiber laser

Observing microvasculature in its native environment provides invaluable information to understand the initiation and development of microcirculatory related diseases. However, the lack of high-resolution three-dimensional (3D) imaging technique hinders in vivo investigation of the microvasculature. Recently, we found that the red blood cells can emit autofluorescence signals with short-wavelength two-photon excitation. In this study, we exploited this property and developed a time-resolved two-photon excitation microscopy system using a home-made 520 nm femtosecond fiber laser as the excitation source. Using this system, we could achieve intravital high-resolution 3D imaging of microvascular network noninvasively. In a mouse tumor model, tumorous blood vessels could be observed and distinguished clearly from the normal vessels.