University of Texas at Austin, Department of Biomedical Engineering, Austin, Texas 78712, USA.
J Biomed Opt. 2011 Feb;16(2):026008. doi: 10.1117/1.3548646.
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.
内源性荧光提供形态、光谱和寿命对比，可以指示组织中的疾病状态。先前的研究表明，双光子自发荧光显微镜（2PAM）可用于对皮肤表面以下约 150μm 深度的上皮组织进行非侵入性、三维成像。我们报告了人舌活检上皮组织的离体 2PAM 图像，深度可达表面以下 370μm。在大于 320μm 深度处，焦外产生的荧光会使图像对比度降低到 1 以下。我们证明，使用传统的 80MHz 重复率超快激光振荡器，160mW 的激光功率（每个脉冲 2nJ）即可达到这些成像深度。为了更好地了解我们在上皮组织中能够达到的最大成像深度，我们研究了具有一系列相关光学特性的组织体模中深度对图像对比度的影响。体模数据与时间分辨蒙特卡罗模拟的估计对比度衰减吻合良好，并且显示出与在人体活检结果中发现的相似的最大成像深度。这项工作表明，与传统的 2PAM 相关的低染色不均匀性（约 20）和大散射系数（约 10mm^-1）限制了上皮组织中最大成像深度为 3 到 5 个平均自由程。
