Department of Physics, University of California, Berkeley, California.
Department of Physics and LSA Biophysics, University of Michigan, Ann Arbor, Michigan.
Biophys J. 2021 Aug 3;120(15):2952-2968. doi: 10.1016/j.bpj.2021.06.025. Epub 2021 Jun 30.
In TIRF microscopy, the sample resides near a surface in an evanescent optical field that, ideally, decreases in intensity with distance from the surface in a pure exponential fashion. In practice, multiple surfaces and imperfections in the optical system and refractive index (RI) inhomogeneities in the sample (often living cells) produce propagating scattered light that degrades the exponential purity. RI inhomogeneities cannot easily be avoided. How severe is the consequent optical degradation? Starting from Maxwell's equations, we derive a first-order perturbative approximation of the electric field strength of light scattered by sample RI inhomogeneities of several types under coherent evanescent field illumination. The approximation provides an expression for the scattering field of any arbitrary RI inhomogeneity pattern. The scattering is not all propagating; some is evanescent and remains near the scattering centers. The results presented here are only a first-order approximation, and they ignore multiple scattering and reflections off the total internal reflection (TIR) surface. For simplicity, we assume that the RI variations in the z direction are insignificant within the depth of the evanescent field and consider only scattering of excitation light, not fluorescence emission light. The general conclusion of most significance from this study is that TIR scattering from a sample with RI variations typical of those on a cell culture alters the effective thickness of the illumination to only ∼50% greater than it would be without scattering. The qualitative surface selectivity of TIR fluorescence is largely retained even in the presence of scattering. Quantitatively, however, scattering will cause a deviation from the incident exponential decay at shorter distances, adding a slower decaying background. Calculations that assume a pure exponential decay will be approximations, and scattering should be taken into account. TIR scattering is only slightly dependent on polarization but is strongly reduced for the highest accessible incidence angles.
在 TIRF 显微镜中，样品位于表面附近的瞬逝光场中，理想情况下，该光场的强度随距表面的距离以纯指数方式减小。实际上，多个表面和光学系统中的不完美以及样品（通常是活细胞）中的折射率（RI）不均匀性会产生传播的散射光，从而降低指数纯度。RI 不均匀性不容易避免。因此，光学退化有多严重？从麦克斯韦方程组出发，我们在相干瞬逝场照明下，针对几种类型的样品 RI 不均匀性，对光散射的电场强度进行了一阶微扰近似。该近似为任意 RI 不均匀性模式的散射场提供了一个表达式。散射并不都是传播的；有些是瞬逝的，并且仍然靠近散射中心。这里给出的结果只是一阶近似，并且它们忽略了多次散射以及全内反射（TIR）表面的反射。为简单起见，我们假设在瞬逝场的深度内 z 方向的 RI 变化可以忽略不计，并且仅考虑激发光的散射，而不是荧光发射光。这项研究的最重要结论是，具有与细胞培养中典型 RI 变化的样品的 TIR 散射会将照明的有效厚度仅改变为比没有散射时增加约 50％。即使存在散射，TIR 荧光的表面选择性也基本保持不变。但是，在定量方面，散射会导致在较短距离处偏离入射指数衰减，从而增加较慢的衰减背景。假设纯指数衰减的计算将是近似值，并且应考虑散射。TIR 散射仅对偏振略有依赖，但对于可达到的最高入射角会大大降低。
