荧光各向异性 - 它是什么？

荧光各向异性是对分子在吸收和发射事件之间的时间上如何改变空间方向的测量。

吸收和发射是相对于激发和发光的电磁波的分子偶极子空间比对的核心指标。为了另一种方式，如果荧光团种群用垂直极化的光激发，那么发射光将根据溶液中旋转的速度保留一些极化。

发射光的去极化量与方向运动的速度有关 - 快速方向运动将导致更多的去极化，反之亦然。

图1。Anisotropy equation.

In order to make use of this information, polarizers are inserted into the excitation light path and the emission light path of a fluorometer. Anisotropy is calculated looking at the ratio of intensities in the above equation, and here IVV indicates the intensity with vertically polarized excitation and vertical polarization on the detected emission.

IVH强调了在发射的激发和水平偏振器上使用垂直偏振器时的强度，而G是光栅因子作为仪器对两个正交矢量取向的差异传输的校正。

Figure 2.使用激发和发射极化器旋转至0度（垂直，V）和90度（水平，H）方向的荧光各向异性实验的描述。

该实验通过首先使用垂直设置的激发偏振层和发射偏振器测量荧光来起作用。然后将强度输入到各向异性方程式中为IVV，然后再用发射偏振器重复测量，然后将其设置为水平方向。将其强度输入为IVH中的方程式。

Figure 3.用于应用各向异性，R和时间分辨各向异性R（T）的一些有用方程。（Lakowicz，2006）（Valeur，2002）

该公式包含两个因子，因为有两个正交取向，可以将VVS矢量的偏转投影到HX和HY上，从而产生两个IVH组件。

极化P的程度通常用于说明仅考虑一个水平分量的二维极化参数。在这种情况下，该公式将不包含ivh的乘数2，其中P代替r。

图4。Time-resolved anisotropy measurement

此外，通过测量HH和HV处的强度，然后将IHV和IHH插入G。各向异性。粘度。

What are the Applications of Fluorescence Anisotropy?

Below it is possible to see a series of equations which can be used for the各向异性结果的分析。The basic anisotropy equation has already been outlined above, but this can also be calculated for whole fluorescence decays, allowing for the calculation of time-resolved anisotropy.

图5。Temperature induced unfolding of BSA protein monitored by fluorescence anisotropy of intrinsic tryptophan residues

Once time-resolved anisotropy has been calculated, it is possible to obtain reorientation time constants and then invoke the Perrin equation and Stokes Einstein Debye equation to gain approximate values for properties like local viscosity, diffusion coefficient and molecular volumes.

These properties relate to crucial information when examining applications like polymer aggregation, protein or molecular binding; or a range of other local environment studies using complex materials and solutions.

在上一个示例中，有可能看到通过荧光各向异性测量的BSA的温度依赖性蛋白展开行为。在这种情况下，已经使用了内在色氨酸残基的各向异性。

荧光动力学的用途是什么？

荧光动力学涉及随着时间的推移观察荧光强度。实际上，这意味着样品会在单个波长上激发，而随着时间的流逝，在单个波长下检测到发射。有时，波长对可用于比率染料，或同时记录基线或背景信息。

图6。左上：硫胺素和HG2+的反应形成硫蛋白原。左下：硫胺素标准的反应速率。右：四个硫胺素标准的荧光强度与硫胺素转化为硫胺素的时间。线性表示每个标准的恒定反应速率。

下图显示了如何使用基于时间的测量值跟随反应速率。在这个特定的示例中，通过改变所用硫胺素的浓度发现硫胺素和汞与形成硫蛋白原的结合的反应速率。这里的每种动力学扫描都表示硫酸盐形成反应的不同反应速率。

通常使用称为停止流量的快速混合配件来测量荧光动力学。停止流量仅在几毫秒内将两个或更多溶液混合在一起，这意味着可以观察到反应或结合，并将其记录到更接近混合时间零而没有任何扩散效应的情况下。

图7。Left: ANS fluorescence intensity vs. time for a stopped flow mixing of BSA and ANS to measure ANS binding to the protein measured on a HORIBA FluoroMax-4. Top Right: Stopped flow fast mixing accessory. Bottom right: Schematic of the stopped flow accessory.

上图显示了称为ANS与蛋白质的荧光团的结合。可以看出，结合时ANS荧光增加，因此在这种情况下，荧光动力学可用于测量结合速率。在这种情况下，ANS与BSA的结合以大约400毫秒的速率发生。

How Can I Control the Sample Temperature on a Fluorometer?

两个非常有用的配件，用于控制荧光计上样品温度Peltier温度控制器和循环浴。在荧光计系统上发现的常规比色杯架具有一对允许液体循环的连接。这些可以连接到循环的水浴，从而在-40°C和70°C之间调节温度。

Circulating baths are useful where a temperature must be set and held during an experiment, but a Peltier temperature controller is a more appropriate choice where samples are highly sensitive to temperature changes or must be measured at different temperatures over a range.

The Peltier controlled cell holder has a much quicker response than a traditional water bath, enabling temperatures within the range of -25 °C to 105 °C to be regulated. These devices are able to achieve a much more precise level of temperature control than a circulating bath which has the tendency to ramp up to a temperature, go over it and then return to it until the target temperature is reached.

As a further option, it is possible to use cryostats and associated mounting kits. These are available for various models of liquid nitrogen and helium cryostats and besides cooling, most of these devices are also able to heat samples up to 500 K and more.

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