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Journal of Biomedical Optics

Tripling the maximum imaging depth with third-harmonic generation microscopy
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Paper Abstract

The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use of longer excitation wavelengths and three-photon-based techniques in nonlinear imaging modalities. This study presents a threefold improvement in maximum imaging depth of ex vivo porcine vocal folds using third-harmonic generation (THG) microscopy at 1552-nm excitation wavelength compared to two-photon microscopy (TPM) at 776-nm excitation wavelength. The experimental, analytical, and Monte Carlo simulation results reveal that THG improves the maximum imaging depth observed in TPM significantly from 140 to 420  μm in a highly scattered medium, reaching the expected theoretical imaging depth of seven extinction lengths. This value almost doubles the previously reported normalized imaging depths of 3.5 to 4.5 extinction lengths using three-photon-based imaging modalities. Since tissue absorption is substantial at the excitation wavelength of 1552 nm, this study assesses the tissue thermal damage during imaging by obtaining the depth-resolved temperature distribution through a numerical simulation incorporating an experimentally obtained thermal relaxation time (τ). By shuttering the laser for a period of 2τ, the numerical algorithm estimates a maximum temperature increase of ∼2°C at the maximum imaging depth of 420  μm. The paper demonstrates that THG imaging using 1552 nm as an illumination wavelength with effective thermal management proves to be a powerful deep imaging modality for highly scattering and absorbing tissues, such as scarred vocal folds.

Paper Details

Date Published: 17 September 2015
PDF: 10 pages
J. Biomed. Opt. 20(9) 096013 doi: 10.1117/1.JBO.20.9.096013
Published in: Journal of Biomedical Optics Volume 20, Issue 9
Show Author Affiliations
Murat Yildirim, The Univ. of Texas at Austin (United States)
Nicholas Durr, The John Hopkins Univ. (United States)
The Univ. of Texas at Austin (United States)
Adela Ben-Yakar, The Univ. of Texas at Austin (United States)

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