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首页> 外文期刊>The journal of physical chemistry, A. Molecules, spectroscopy, kinetics, environment, & general theory >Another Treatment of Fluorescence Polarization Microspectroscopy and Imaging
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Another Treatment of Fluorescence Polarization Microspectroscopy and Imaging

机译：荧光偏振光谱和成像的另一种治疗方法

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We here discuss a general (symmetry adapted) treatment for one-photon-excitation time-resolved fluorescence polarization microspectroscopy (TRFPM) at combined wide-angular excitation and detection apertures that correctly couples the principles of the optics of objective lenses with the principles of fluorescence spectroscopy with polarized light. The treatment is unified in the sense that it covers the electromagnetic description of focusing a linearly polarized beam of exciting light (diffraction theory, DT) and the description of the same problem in terms of the meridional plane properties (MPP) of the objective lenses (geometrical optics). It is shown that both approaches are quantitatively equivalent from the point of view of the polarization effects in typical TRFPM experiments on linear absorbers, despite the fact that in the MPP treatment the region of focus is treated as a pointlike object, while in the DT method the region of focus is characterized by a threedimensional (3D) inhomogeneous electromagnetic field distribution, of generally ellipsoidal polarization at different points of the focus. This finding is essentially important from the point of view of the experimental practice because the MPP treatment is based on two very simple trigonometric expressions, in evident contrast to the DT method, in which the high-aperture focusing is described in terms of three complicated 3D integrals involving the Bessel functions of the first kind. A few words of comment are added on a similar problem in the case of nonlinear one-photon absorbers (e.g., chiral fluorophores). We discuss the synthetic fluorescence decays for the wide-field- and evanescent-wave-excitation confocal (or wide-field) detection fluorescence polarization microspectroscopy and imaging, which indicate the right experimental protocols for the kinetic and dynamic fluorescence polarization microspectroscopic studies. The manifestations of the effects resulting from the application of the wide-angular excitation and/or detection apertures are displayed and discussed in a systematic way. A few words of comment are added on the application of the symmetry adapted calibration (SAC) method to TRFPM experiments. A very important aim of this article is to provide a correct and more complete description of fluorescence polarization microspectroscopy and imaging of macroscopically isotropic media (i.e., solutions, solutions of labeled macromolecules, membrane suspensions, or biological cells), that can be immediately applied in the experimental practice in the life and medical sciences and also in different areas of nano(bio)technology.
机译：我们在这里讨论在组合的广角激发和检测孔径下将单光子激发时间分辨荧光偏振光谱（TRFPM）进行的常规（对称适应）处理，该处理可以正确地将物镜的光学原理与荧光的原理相结合。偏振光光谱。这种处理是统一的，因为它涵盖了聚焦线性偏振激发光束的电磁描述（衍射理论，DT）和关于物镜的子午面特性（MPP）的相同问题的描述（几何光学）。从典型TRFPM实验中线性吸收体的极化效应来看，尽管在MPP处理中将焦点区域视为点状对象，而在DT方法中，这两种方法在数量上是等效的焦点区域的特征是在焦点的不同点处具有大致椭圆形极化的三维（3D）不均匀电磁场分布。从实验实践的角度来看，这一发现非常重要，因为MPP处理基于两个非常简单的三角表达式，这与DT方法形成鲜明对比的DT方法不同，后者是根据三种复杂的3D描述高光圈聚焦的涉及第一类Bessel函数的积分。对于非线性单光子吸收剂（例如手性荧光团），在类似问题上也加了一些评论。我们讨论了在宽场和e逝波激发共焦（或宽场）检测荧光偏振显微光谱和成像中合成的荧光衰减，这为动力学和动态荧光偏振显微光谱研究指明了正确的实验方案。通过广角激发和/或检测孔的应用所产生的影响的表现方式以系统的方式显示和讨论。在将对称适应校准（SAC）方法应用于TRFPM实验时，添加了一些评论。本文的一个非常重要的目的是为荧光偏振光谱技术和宏观各向同性的介质（即溶液，标记的大分子溶液，膜悬浮液或生物细胞）成像提供正确，更完整的描述，可以立即将其应用于生命和医学科学以及纳米（生物）技术不同领域的实验实践。

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