title: Maximizing fluorescence collection efficiency in multiphoton microscopy.
abstract: Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% - 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm.
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06511, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Zinter
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Levene
mesh_terms: D000818:Animals; D003198:Computer Simulation; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009010:Monte Carlo Method; D009474:Neurons / Q000473:pathology*; D055095:Optics and Photonics / Q000379:methods; D019047:Phantoms, Imaging; D012542:Scattering, Radiation; D013598:Systems Theory