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Proceedings Paper

Strategies for improving depth-penetration of two-photon imaging in vivo
Author(s): Martin Oheim; Emmanuel Beaurepaire; Jerome Mertz; Serge Charpak
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Paper Abstract

We investigate tissue and instrument parameters affecting the penetration depth in two-photon microscopy. We show that the temporal redistribution of the same average power into fewer pulses of higher peak energy by means of a regenerative amplifier results in an increase in excitation depth by approximately 2-3 scattering mean free paths. We then measure the excitation scattering mean free path in vitro, using rat brain slices, as a function of the excitation wavelength and tissue age. We find that young-animal tissue (< P18) is two-fold less scattering than adult tissue (P90). We quantify the fall-off of the collected fraction of generated fluorescence in a backward detection geometry, in vivo. At large depths, we observe that the collected fraction scales as the angular acceptance squared (related to the effective field-of-view) of the detection optics. Matching the angular acceptance of the detection optics to that of the objective (63X NA-0.90) results in a factor 3-4 of the collected fluorescence. The collection efficiency can be further increased (10X) by using an objective with large field-of-view and high numerical aperture (20X NA-0.95). These gains translate into approximately 120 micrometers additional depth penetration when working in the rat brain in vivo with a standard Ti:sapphire source.

Paper Details

Date Published: 2 November 2001
PDF: 7 pages
Proc. SPIE 4431, Photon Migration, Optical Coherence Tomography, and Microscopy, (2 November 2001); doi: 10.1117/12.447410
Show Author Affiliations
Martin Oheim, Ecole Superieure de Physique et Chimie Industrielles (France)
Emmanuel Beaurepaire, Ecole Superieure de Physique et Chimie Industrielles (France)
Jerome Mertz, Ecole Superieure de Physique et Chimie Industrielles (France)
Serge Charpak, Ecole Superieure de Physique et Chimie Industrielles (France)

Published in SPIE Proceedings Vol. 4431:
Photon Migration, Optical Coherence Tomography, and Microscopy
Stefan Andersson-Engels; Michael F. Kaschke, Editor(s)

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