Maximizing fluorescence collection efficiency in multiphoton microscopy.

Opt Express

Department of Biomedical Engineering, Yale University, New Haven, CT 06511, USA.

Published: August 2011

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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482884PMC
http://dx.doi.org/10.1364/OE.19.015348DOI Listing

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