title: Tripling the maximum imaging depth with third-harmonic generation microscopy.
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.
authors.affiliation: The University of Texas at Austin, Department of Mechanical Engineering, 204 East Dean Keeton Street, Stop C2200, Austin, Texas 78712, United States.
authors.forename: Murat
authors.initials: M
authors.lastname: Yildirim
authors.affiliation: The John Hopkins University, Department of Biomedical Engineering, 3400 North Charles Street, Baltimore, Maryland 21218, United StatescThe University of Texas at Austin, Department of Biomedical Engineering, 107 West Dean Keeton Street, Stop C0800, Austin.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Durr
authors.affiliation: The University of Texas at Austin, Department of Mechanical Engineering, 204 East Dean Keeton Street, Stop C2200, Austin, Texas 78712, United StatescThe University of Texas at Austin, Department of Biomedical Engineering, 107 West Dean Keeton Street, Stop.
authors.forename: Adela
authors.initials: A
authors.lastname: Ben-Yakar
mesh_terms: D000465:Algorithms; D000818:Animals; D003198:Computer Simulation; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007834:Lasers; D008853:Microscopy / Q000379:methods*; D009010:Monte Carlo Method; D013552:Swine; D013696:Temperature; D014827:Vocal Cords / Q000502:physiology