Two-photon excited hemoglobin fluorescence provides contrast mechanism for label-free imaging of microvasculature in vivo.

Direct visualization of microvasculature provides significant insights in microcirculation and critically impacts the diagnosis and treatment of microcirculatory diseases. Recently, we discovered that the high-energy Soret fluorescence of hemoglobin peaked at 438 nm with an extremely short lifetime becomes strongly visible under two-photon excitation. Based on the distinct spectral and temporal characteristics of hemoglobin fluorescence, we demonstrated that two-photon fluorescence microscopy could become a powerful tool for label-free in vivo imaging of microvasculature in tissue.