Process Analysis and Technology, Reutlingen Research Institute, Reutlingen University, Alteburgstr. 150, 72762, Reutlingen, Germany.
Institute of Physical and Theoretical Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076, Tübingen, Germany.
Anal Bioanal Chem. 2021 Nov;413(28):7093-7106. doi: 10.1007/s00216-021-03678-w. Epub 2021 Oct 1.
The article analyzes experimentally and theoretically the influence of microscope parameters on the pinhole-assisted Raman depth profiles in uniform and composite refractive media. The main objective is the reliable mapping of deep sample regions. The easiest to interpret results are found with low magnification, low aperture, and small pinholes. Here, the intensities and shapes of the Raman signals are independent of the location of the emitter relative to the sample surface. Theoretically, the results can be well described with a simple analytical equation containing the axial depth resolution of the microscope and the position of the emitter. The lower determinable object size is limited to 2-4 μm. If sub-micrometer resolution is desired, high magnification, mostly combined with high aperture, becomes necessary. The signal intensities and shapes depend now in refractive media on the position relative to the sample surface. This aspect is investigated on a number of uniform and stacked polymer layers, 2-160 μm thick, with the best available transparency. The experimental depth profiles are numerically fitted with excellent accuracy by inserting a Gaussian excitation beam of variable waist and fill fraction through the focusing lens area, and by treating the Raman emission with geometric optics as spontaneous isotropic process through the lens and the variable pinhole, respectively. The intersectional area of these two solid angles yields the leading factor in understanding confocal (pinhole-assisted) Raman depth profiles. Spearfishing is a well-known example of the effects of refraction at the boundary between two index-mismatched media. The object G is seen, due to refraction, as G from the angle β (without knowing the depth position). The real position is obtained under the angle α. In a microscope (see inset), index mismatch deforms the image point of G into an image line. The pinhole substantially reduces deformations and allows the determination of the position of the point emitter G. (Cartoon designed by Sofia Anker).
本文从实验和理论两方面分析了显微镜参数对均匀和复合折射介质中针孔辅助拉曼深度轮廓的影响。主要目标是可靠地绘制深层样品区域的图谱。使用低倍放大率、低孔径和小孔时，可获得最易于解释的结果。在此，拉曼信号的强度和形状与发射器相对于样品表面的位置无关。从理论上讲，这些结果可以用一个简单的解析方程很好地描述，该方程包含显微镜的轴向深度分辨率和发射器的位置。可确定的最小物体尺寸下限为 2-4μm。如果需要亚微米分辨率，则需要高倍放大率，通常与高孔径结合使用。在折射介质中，信号强度和形状现在取决于相对于样品表面的位置。在许多具有最佳透明度的 2-160μm 厚的均匀和堆叠聚合物层上对此进行了研究。实验深度轮廓通过插入可变腰宽和填充分数的高斯激发光束，通过聚焦透镜区域，并通过几何光学分别将拉曼发射处理为通过透镜和可变针孔的各向同性自发过程，对其进行了数值拟合，具有出色的精度。这两个立体角的交叉区域是理解共焦（针孔辅助）拉曼深度轮廓的关键因素。射鱼叉捕鱼是两种折射率不匹配的介质边界折射效果的一个众所周知的例子。由于折射，物体 G 从角度β（不知道深度位置）被看到为 G。真实位置是在角度α下获得的。在显微镜下（参见插图），折射率失配对 G 的像点进行变形，成为像线。针孔大大减少了变形，并允许确定点发射器 G 的位置。（由 Sofia Anker 设计的卡通画）。
