Dunn A K, Wallace V P, Coleno M, Berns M W, Tromberg B J
Laser Medical and Microbeam Program, Beckman Laser Institute, University of California, Irvine, California 92612, USA.
Appl Opt. 2000 Mar 1;39(7):1194-201. doi: 10.1364/ao.39.001194.
A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..
开发了一个数值模型，以模拟组织光学特性、物镜数值孔径（N.A.）和仪器性能对浑浊样品双光子激发荧光成像的影响。将模型数据与在类似组织的散射体模中对荧光微球的测量结果进行了比较。我们的结果表明，测量到的双光子激发信号随焦深增加呈指数衰减。总体衰减常数是激发和发射波长处吸收和散射参数的函数。结果表明，除了g>0.95外，双光子荧光的产生与散射各向异性g无关。收集到最大信号的数值孔径随深度变化，不过直到焦平面进入样品超过两个散射平均自由程时才会看到这种效应。总体而言，测量结果和模型结果表明，双光子显微镜中的分辨率仅取决于向焦体积传递足够弹道光子密度的能力。结果我们表明，在软组织中通常遇到的范围内，双光子显微镜的横向分辨率在很大程度上不受组织光学特性的影响，尽管最大成像深度强烈依赖于吸收系数、散射系数、散射各向异性和物镜数值孔径。
