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首页> 外文期刊>The journal of physical chemistry, A. Molecules, spectroscopy, kinetics, environment, & general theory >Fluorescence Polarization Spectroscopy at Combined High-Aperture Excitation and Detection: Application to One-Photon-Excitation Fluorescence Microscopy
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Fluorescence Polarization Spectroscopy at Combined High-Aperture Excitation and Detection: Application to One-Photon-Excitation Fluorescence Microscopy

机译：组合的高光圈激发和检测的荧光偏振光谱：在单光子激发荧光显微镜中的应用。

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A problem of the one-photon-excitation fluorescence polarization spectroscopy of macroscopically isotropic media, in the case of combined high-aperture excitation and detection, is considered and described in a spherical representation. The case of inhomogeneous intensity distribution in the cross-section of the parallel beam of exciting light, which is converted by an objective lens into inhomogeneous radial distribution of the intensity of the focused exciting light, is also taken into account. The obtained formalism is adapted to the description of confocal fluorescence polarization microscopy. It is shown that the total and magic-angle-detected fluorescence decays do not solely represent the kinetic evolution of the excited-state because of the contribution of the dynamic evolution of photoselected fluorophores. The time-evolution of emission anisotropy is nonexponential. The outlined theory predicts that the total and magic-angle-detected fluorescence decays solely represent the kinetic fluorescence decay, and thereby, the emission anisotropy becomes an (multi)-exponential function of time for the excitation-detection cone half-angles not higher than about 15-20°. The initial values of the emission anisotropy are not modified by the application of the excitation-detection apertures if the cone half-angles do not exceed 10-15°. The histograms of unpolarized fluorescence, calculated from the parallel and the perpendicular components of polarized fluorescence, detected at the excitation-detection cones wider than about 65° solely represent the kinetic fluorescence decay. At such conditions, the microscope objective operates like an "integrating sphere". The calibration method, which is based on a general (symmetry adapted) formula describing fluorescence polarization experiments on macroscopically isotropic samples, is discussed. This method enables the analysis of all fluorescence polarization experiments without the necessity of considering the expressions for polarized fluorescence decays relating to a particular experimental case of interest. With this method, any commercially available microscope objective can be calibrated, and its optical properties can be precisely verified. The application of the outlined theory to different fluorescence spectroscopy techniques is indicated. The expressions derived for confocal fluorescence polarization microscopy can be employed in the numerical analysis of the data recovered from the photochemical bioimaging.
机译：在结合高孔径激发和检测的情况下，以球形表示形式考虑和描述了宏观各向同性介质的单光子激发荧光偏振光谱问题。还考虑了在激发光的平行光束的横截面中强度分布不均匀的情况，该情况由物镜转换为聚焦激发光的强度的不均匀径向分布。所获得的形式适合于共焦荧光偏振显微镜的描述。结果表明，总的和魔术角检测到的荧光衰减并不仅仅代表激发态的动力学演化，这是由于光选择荧光团的动态演化所致。发射各向异性的时间演化是非指数的。概述的理论预测，总的和魔角​​检测到的荧光衰减仅表示动能荧光衰减，因此，对于激发-检测锥半角不大于，发射各向异性成为时间的（多）指数函数。约15-20°。如果锥形半角不超过10-15°，则发射各向异性的初始值不会通过激励检测孔的应用而改变。由偏振荧光的平行和垂直分量计算得到的未偏振荧光的直方图，在大于约65°的激发检测锥处检测到，仅表示动力学荧光衰减。在这种条件下，显微镜物镜的工作就像一个“积分球”。讨论了基于一般（对称适应）公式的校准方法，该公式描述了宏观各向同性样品的荧光偏振实验。该方法能够分析所有荧光偏振实验，而无需考虑与特定实验案例有关的偏振荧光衰减的表达式。使用这种方法，可以校准任何市售的显微镜物镜，并且可以精确地验证其光学特性。指出了概述的理论在不同荧光光谱技术中的应用。用于共聚焦荧光偏振显微镜的表达式可用于对从光化学生物成像中回收的数据进行数值分析。

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