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Journal of Biomedical Optics • Open Access

Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues
Author(s): Nicholas J. Durr; Adela Ben-Yakar; Christian T. Weisspfennig; Benjamin A. Holfeld

Paper Abstract

Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼20) and large scattering coefficient (∼10 mm−1) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue.

Paper Details

Date Published: 1 February 2011
PDF: 13 pages
J. Biomed. Opt. 16(2) 026008 doi: 10.1117/1.3548646
Published in: Journal of Biomedical Optics Volume 16, Issue 2
Show Author Affiliations
Nicholas J. Durr, The Univ. of Texas at Austin (United States)
Adela Ben-Yakar, The Univ. of Texas at Austin (United States)
Christian T. Weisspfennig, The Univ. of Texas at Austin (United States)
Benjamin A. Holfeld, The Univ. of Texas at Austin (United States)

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