title: Frequency MultiplexedIn VivoMultiphoton Phosphorescence Lifetime Microscopy.
abstract: 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 depthin 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.
authors.affiliation: School of Applied and Engineering Physics, Cornell University, 212 Clark Hall, Ithaca, NY 14853. Department of Electrical Engineering, University of Notre Dame, 275 Fitzpatrick Hall, Notre Dame, IN 46556.
authors.forename: Scott S
authors.initials: SS
authors.lastname: Howard
authors.forename: Adam
authors.initials: A
authors.lastname: Straub
authors.forename: Nicholas
authors.initials: N
authors.lastname: Horton
authors.forename: Demirhan
authors.initials: D
authors.lastname: Kobat
authors.forename: Chris
authors.initials: C
authors.lastname: Xu