Frequency Multiplexed In Vivo Multiphoton Phosphorescence Lifetime Microscopy

Multiphoton microscopy (MPM) is widely used for optical sectioning deep in scattering tissue, in vivo [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 experimentally demonstrate a parallel-excitation/parallel collection MPM-PLIM system that increases 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.