Maximizing fluorescence collection efficiency in multiphoton microscopy

Opt Express. 2011 Aug 1;19(16):15348-62. doi: 10.1364/OE.19.015348.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Computer Simulation
  • Equipment Design
  • Image Processing, Computer-Assisted
  • Mice
  • Microscopy, Fluorescence / methods*
  • Monte Carlo Method
  • Neurons / pathology*
  • Optics and Photonics / methods
  • Phantoms, Imaging
  • Scattering, Radiation
  • Systems Theory