Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy

Multiphoton microscopy (MPM) is widely used in vivo for optical sectioning deep inside scattering tissue 1 , 2 . Phosphorescence lifetime imaging microscopy (PLIM) 3 is a powerful technique for obtaining biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching 4 , 5 . Point-measurement PLIM 6 of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM 7 , 8 . However, the maximum fluorescence generation rate, which is inversely proportional to the phosphorescence lifetime, fundamentally limits PLIM pixel rates. Here, we demonstrate experimentally a parallel-excitation/parallel-collection MPM–PLIM system that increases the pixel rate by a factor of 100 compared with conventional configurations, while simultaneously acquiring lifetime and intensity images at depth in vivo . Full-frame, three-dimensional, in vivo PLIM imaging of phosphorescent quenching dye is presented for the first time and defines a new platform for biological and medical imaging. A parallel implementation of multifocal multiphoton modulation microscopy allows simultaneous phosphorescent lifetime and intensity imaging in vivo at speeds 100 times faster than conventional configurations. Three-dimensional imaging of a phosphorescent quenching dye is also presented.