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- Title
Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy.
- Authors
Howard, Scott S.; Straub, Adam; Horton, Nicholas G.; Kobat, Demirhan; Xu, Chris
- Abstract
Multiphoton microscopy (MPM) is widely used in vivo for optical sectioning deep inside scattering tissue. Phosphorescence lifetime imaging microscopy (PLIM) is a powerful technique for obtaining biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching. Point-measurement PLIM of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM. 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.
- Subjects
MULTIPHOTON excitation microscopy; FLUORESCENCE resonance energy transfer; PHOSPHORESCENCE; FLUORIMETRY; DIAGNOSTIC imaging equipment
- Publication
Nature Photonics, 2013, Vol 7, Issue 1, p33
- ISSN
1749-4885
- Publication type
Article
- DOI
10.1038/nphoton.2012.307