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Applied Optics

Applied Optics


  • Editor: Joseph N. Mait
  • Vol. 49, Iss. 8 — Mar. 10, 2010
  • pp: 1472–1479

Calculated two-photon fluorescence correction factors for reflective scan engines

Galiya Sharafutdinova, John Holdsworth, and Dirk van Helden  »View Author Affiliations

Applied Optics, Vol. 49, Issue 8, pp. 1472-1479 (2010)

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Excitation laser spatial and temporal characteristics at the objective focal point are critical to the performance of two-photon scanning microscopes. Optical aberrations in scanning systems increase the microscope objective focal spot area and introduce pulse time broadening in the deflected beam, resulting in degradation of two-photon-induced fluorescence across the scan field. The geometrical pulse broadening is investigated for what is believed to be the first time and then combined with a focused spot area to provide a normalized two-photon fluorescence intensity correction factor. This factor, calculated using OSLO optical software, is compared for four reflective scan engines and allows compensation of the detected signal with position across the scan field. This new metric highlights that a parabolic mirror afocal relay exhibits superior performance as a reflective scan engine for two-photon scanning microscopy.

© 2010 Optical Society of America

OCIS Codes
(170.5810) Medical optics and biotechnology : Scanning microscopy
(180.5810) Microscopy : Scanning microscopy
(080.4035) Geometric optics : Mirror system design
(080.4228) Geometric optics : Nonspherical mirror surfaces
(080.6755) Geometric optics : Systems with special symmetry

Original Manuscript: November 30, 2009
Revised Manuscript: January 31, 2010
Manuscript Accepted: February 4, 2010
Published: March 9, 2010

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Vol. 5, Iss. 7 Virtual Journal for Biomedical Optics

Galiya Sharafutdinova, John Holdsworth, and Dirk van Helden, "Calculated two-photon fluorescence correction factors for reflective scan engines," Appl. Opt. 49, 1472-1479 (2010)

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