Fluorescence photobleaching recovery spectroscopy in a dye doped nematic liquid crystal
Fluorescence photobleaching recovery spectroscopy in a dye doped nematic liquid crystal
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染料掺杂向列液晶中的荧光光漂白恢复光谱
DOI：
10.1103/physreve.59.1860
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
P. Etchegoin
中科院分区：
文献类型：
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作者：
P. Etchegoin
荧光标记的蛋白质或葡聚糖在细胞或生物相关流体内的扩散动力学的原位研究是生物光子学@1，2#领域的惯例。通常，与特定大分子或蛋白质结合的染料被用作示踪剂，以跟踪后者的特定动力学。染料可以通过通常由显微镜物镜聚焦的外部激光器共振泵浦。标记分子发出的荧光可以通过同一物镜收集，随后进行过滤和检测。如果染料的光漂白发生在物镜的聚焦区域内，则在激光照射的早期阶段观察到荧光信号的衰减。由光漂白产生的衰减时间取决于输入功率、特定激光波长和染料吸收光谱的特性。另一方面，如果激光器关闭给定的时间段，则当激光照射恢复时，可以观察到荧光信号的恢复，因为新分子从相邻区域扩散到激光光斑的焦点体积中。以这种方式，可以研究标记分子本身的扩散过程。事实上，生物分子的扩散是细胞生物学@3#中最基本也是最重要的机制之一，并且可以通过这种方式进行显微镜研究。术语荧光光漂白恢复光谱~FPRS！已经被创造出来作为这种方法的一般描述。该技术根据具体实验的细节有不同的变化。特别是，照明光学器件的几何方面和监测荧光发射的成像方法-正常，共焦等！确定正在考虑的实验类型。最近，除了FPRS之外，还提出了使用双光子扫描显微镜@4#作为用于创建光漂白分子的平坦的、几乎二维的分布的方法，允许在扩散发生之前更好地控制初始条件。液晶~LC的！是具有内在长程取向有序性和复杂流体力学性质的复杂流体。染料分子可以在LC内稀释，以这种方式利用液晶相的固有分子有序性，并且变得沿沿着特定方向取向。Heilmeier，Castellano和Zanoni将染料的诱导宏观有序称为宾主相互作用，他们第一次在6 #下观察到它。由于染料的吸收通常是非常各向异性的，染料掺杂的LC是理想的系统，其中二向色性可以通过外部参数如温度和磁场或电场来控制。此外，染料添加到可调液晶已被证明是几个非常有趣和复杂的非线性光学性质的来源。特别是，观察到增强的光学扭矩@7，8 #，不寻常的再取向动力学在超短激光照明@9#，和永久全息记录的图案@10 #是这些系统的丰富的光学特性的一些不完整的例子。从应用的角度来看，染料掺杂液晶材料似乎是一种很有前途的材料，可用于电光器件，在可见光区具有增强的对比度@11#。染料在液晶中的扩散过程迄今为止研究得很少。在这篇论文中，我们将集中研究染料在有向和无向液晶基质中的扩散。特别是，FPRS的应用，温度的影响，并通过荧光发射的数字成像的扩散过程的直接视图将被证明。染料在有序的双相液晶盒内的扩散以各向异性的方式发生，这确保了二色性的长期保持。利用双光束FPRS技术可以直接观察到光漂白染料分子的扩散过程。光漂白和扩散之间的相互作用将被明确显示，以及温度对平面取向的液晶盒的影响，其中染料分子被局部光漂白。此外，我们表明，可以使用拉曼信号而不是荧光信号来研究透明物质（例如线虫二元混合物）的相互扩散，其中不能使用固有荧光。该文件的组织如下：第二节介绍了即将进行的实验，样品制备，解释的数据，和实验结果的实验装置。必要的理论背景也提供了第二节。第二，只要它与数据的解释有关。最后，在SEC。第三部分，对本文的研究进行了简要的讨论，并得出了一些结论。
The in situ study of the diffusion dynamics of fluorescently tagged proteins or dextrants within cells or biologically relevant fluids is a customary practice in the field of biophotonics @1,2#. Normally, dyes which bind to specific macromolecules or proteins are employed as tracers to follow the particular dynamics of the latter. The dyes can be resonantly pumped by an external laser which is usually focused by a microscope objective. The fluorescence emitted by the tagged molecules can be collected by the same objective, to be subsequently filtered and detected. If photobleaching of the dyes takes place within the focal region of the objective, a decay of the fluorescence signal is observed in the early stages of laser illumination. The decay time produced by photobleaching depends on the input power, specific laser wavelength, and peculiarities of the absorption spectrum of the dyes. On the other hand, if the laser is turned off for a given period of time, a recovery of the fluorescence signal can be observed when the laser illumination is reinstated, for diffusion of new molecules takes place from the neighboring regions into the focal volume of the laser spot. In this manner, the diffusion process of the tagged molecules themselves can be studied. Diffusion of biomolecules is, in fact, one of the most basic an important mechanisms in cell biology @3#, and can be microscopically studied in this manner. The term fluorescence photobleaching recovery spectroscopy ~FPRS! has been coined as a general description of this method. The technique has different variations according to the details of the specific experiment. In particular, the geometrical aspects of the illuminating optics and the imaging method to monitor the fluorescence emission ~normal, confocal, etc.! determine the type of experiment under consideration. Very recently, the usage of two-photon scanning microscopy in addition to FPRS has been proposed @4# as a method for creating a flat, almost two-dimensional, distribution of photobleached molecules, allowing a better control of the initial conditions before diffusion takes place. Liquid crystals ~LC’s ! are complex fluids with intrinsic long range orientational order and intricate hydrodynamic properties @5#. Dye molecules can be diluted within a LC to take advantage, in this manner, of the intrinsic molecular order of the liquid crystalline phases, and become oriented along particular directions. The induced macroscopic order of the dyes was called guest-host interaction by Heilmeier, Castellano, and Zanoni, who observed it for the first time@6#. Since the absorption of the dyes is normally very anisotropic, dye doped LC’s are ideal systems where dichroism can be controlled by external parameters like temperature and magnetic or electric fields. Furthermore, the addition of dyes to nematic liquid crystals has been shown to be the source of several very interesting and complex nonlinear optical properties. In particular, the observation of enhanced optical torques @7,8#, unusual reorientation dynamics upon ultrashort laser illumination @9#, and permanent holographic recording of patterns @10# are a few incomplete examples of the rich optical properties of these systems. From the viewpoint of applications, dye doped LC’s seem to be promising substances for electro-optic devices with enhanced contrast ratios in the visible @11#. The diffusion process of dyes in LC’s has heretofore been poorly studied. In this paper, we shall concentrate on the study of dye diffusion through both oriented and unoriented nematic liquid crystal hosts. In particular, the application of FPRS, the effect of temperature, and a direct view into the diffusion process by means of digital imaging of the fluorescence emission will be demonstrated. Dye diffusion within an ordered nematic LC cell takes place in an anisotropic manner, which ensures the long term preservation of the dichroism. A direct view into the diffusion process of photobleached dye molecules will be shown by virtue of a twobeam FPRS technique. The interplay between photobleaching and diffusion will be explicitly shown, as well as the effect of temperature on a planar oriented LC cell where dye molecules are locally photobleached. Moreover, we show that Raman rather than fluorescence signals could be used to study the interdiffusion of transparent substances like binary mixtures of nematogens, where intrinsic fluorescence cannot be used. The paper is organized as follows: Section II presents the experimental setup for the forthcoming experiments, the sample preparation, a description of the interpretation of the data, and the experimental findings. The necessary theoretical background is also supplied in Sec. II, whenever it is relevant for the interpretation of the data. Finally, in Sec. III, a brief discussion is presented and a few conclusions are