Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues
Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues
复制标题
DOI：
10.1117/1.3548646
复制
发表时间：
2011-02-01
影响因子：
3.5
通讯作者：
Ben-Yakar, Adela
中科院分区：
文献类型：
--
作者：
Durr, Nicholas J.;Weisspfennig, Christian T.;Ben-Yakar, Adela
内源荧光提供形态、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 150 μm 的上皮组织进行无创三维成像。我们报告了来自人舌活检的上皮组织的离体 2PAM 图像，其深度为表面以下 370 μm。当深度超过 320 μm 时，焦点体积外部产生的荧光会将图像对比度降低到 1 以下。我们证明，使用传统 80 MHz 重复率超快激光振荡器的 160 mW 激光功率（每脉冲 2 nJ）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可以实现的最大成像深度，我们研究了具有一系列相关光学特性的组织模型中的图像对比度作为深度的函数。模型数据与时间分辨蒙特卡罗模拟估计的对比度衰减非常吻合，并显示出与人体活检结果相似的最大成像深度。这项工作表明，与传统 2PAM 相关的低染色不均匀性（类似于 20）和大散射系数（类似于 10 mm(-1)）将最大成像深度限制为上皮组织深处的 3 至 5 个平均自由散射长度。 (C) 2011 年光电仪器工程师协会 (SPIE)。 [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 mu m beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 mu m below the surface. At greater than 320 mu m 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 (similar to 20) and large scattering coefficient (similar to 10 mm(-1)) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3548646]