title: Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores.
abstract: It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.
authors.affiliation: Department of Chemistry, Columbia University, New York, New York 10027, USA.
authors.forename: Zhixing
authors.initials: Z
authors.lastname: Chen
authors.forename: Lu
authors.initials: L
authors.lastname: Wei
authors.forename: Xinxin
authors.initials: X
authors.lastname: Zhu
authors.forename: Wei
authors.initials: W
authors.lastname: Min
mesh_terms: D000465:Algorithms; D005456:Fluorescent Dyes / Q000528:radiation effects*; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008027:Light; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes