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Nat Photonics. 2013 Jan 1;7(1):33-37. Epub 2012 Dec 16.

Frequency Multiplexed In Vivo Multiphoton Phosphorescence Lifetime Microscopy.

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  • 1School 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.


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.

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