Epifluorescence collection in two-photon microscopy.
We present a simple model to describe epifluorescence collection in two-photon microscopy when one images in a turbid slab with an objective. Bulk and surface scattering determine the spatial and angular distributions of the outgoing fluorescence photons at the slab surface, and geometrical optics determines how efficiently the photons are collected. The collection optics are parameterized by the objective's numerical aperture and working distance and by an effective collection field of view. We identify the roles of each of these parameters and provide simple rules of thumb for the optimization of the epifluorescence collection efficiency. Analytical results are corroborated by Monte Carlo simulation.
Beaurepaire
Emmanuel
E
Laboratoire de Neurophysiologie et Nouvelles Microscopies, Institut National de Santé et de Recherche Médicale EPI 00-02, Ecole Supérieure de Physique et Chimie Industrielle, Paris, France.
Mertz
Jerome
J
D003198:Computer Simulation; D004058:Diffusion; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D009010:Monte Carlo Method; D055095:Optics and Photonics; D017785:Photons; D012542:Scattering, Radiation