Quantitative microscopy based on photon statistics (PhotoQuant)

定量信息是在分子尺度上理解生物体复杂内部运作的关键。因此，需要能够精确确定构成结构或参与反应的不同分子物种的拷贝数的方法。理想情况下，这种方法应该提供绝对数，能够在活体标本中进行原位测量的微创方法，以及非破坏性和快速的，以允许重复量化动态复合体。基于荧光显微镜的分子计数有不同的方法，然而，现有的方法都不能同时满足上述所有要求。我们已经证明，衍射受限结构中的单个荧光发射体可以通过记录和分析该结构的光子发射统计数据(按光子统计-COPS计数)进行高精度计数。到目前为止，COPS方法能够在体外实现高时间分辨率和高计数范围的无损测量。为了实现生物分子的原位计数，我们建议扩展COPS方法以考虑动态生物样本日益增加的复杂性。首先，我们计划扩展目前实施的单色点测量方案，以允许基于脉冲交错激发方案的双色同时成像和量化。这些工作将包括修改现有的显微镜设置、显微镜控制和数据读出电子设备以及调整数据分析程序。然后，我们希望通过样本优化、对特定结构的测量和模拟来解决非理想样本的光子统计测量的特殊要求，即背景荧光增强和单个发射体之间的潜在相互作用。然后，所建立的方法将被用于研究两个生物模型系统，即存在HIV效应蛋白Nef时的T细胞受体簇和干扰素α受体及其相关激酶的复杂形成。目标蛋白的荧光标记将使用HaloTag等酶标记实现，而翻译后修饰的蛋白将通过单抗标记。为了将计数的荧光团的数量与目标分子的潜在数量联系起来，将对实现的标记程度进行彻底的表征。最后，我们希望探索将基于点的COPS扩展到使用相机探测器的成像COPS的技术可能性。这将涉及一项基于EMCCD相机的原理证明研究，以在成像模式下表征定义的样品，以及建立空间分辨光子统计数据评估的理论模型。此外，我们还希望评估替代检测方案的潜力，例如用于成像具有更高时间分辨率的COPS的APD阵列。

Quantitative information is key to understand the complex inner workings of living organisms on the molecular scale. Methods that are able to precisely determine copy numbers of different molecular species making up a structure or involved in a reaction are thus required. Ideally, such methods should provide absolute numbers, be minimally invasive enabling in situ measurements in living specimen as well as non-destructive and fast to allow for repeated quantification of dynamic complexes. Different approaches for molecular counting based on fluorescence microscopy exist, however, none of the existing methods is able to fulfill all of the aforementioned requirements at once. We have shown that individual fluorescent emitters in a diffraction-limited structure can be counted with high precision by recording and analyzing photon emission statistics from that structure (counting by photon statistics - CoPS). So far, the CoPS method enables non-destructive measurements with high time resolution and high counting range in vitro.To enable in situ counting of biomolecules, we propose to extend the CoPS methodology to account for the increased complexity of dynamic biological specimen. First, we plan to extend the currently implemented one-color point measuring scheme to allow two-color simultaneous imaging and quantification based on a pulsed interleaved excitation scheme. These works will include modifying an existing microscope setup, the microscope control and data readout electronics as well as adapting the data analysis routine. We then want to address special requirements of photon statistics measurements on non-ideal samples, i.e. elevated background fluorescence and potential interaction between individual emitters by means of sample optimization, measurements on defined structures and simulations.The established approaches will then be used to investigate two biological model systems, namely the T-cell receptor cluster upon presence of the HIV effector protein Nef and the complex formation of interferon alpha receptors and their associated kinases. Fluorescent labeling of the target proteins will be achieved using enzyme tags such as HaloTag while post-translationally modified proteins will be labeled via monoclonal antibodies. To relate the number of counted fluorophores to the underlying number of target molecules, a thorough characterization of the achieved degree of labeling will be performed.Finally, we want explore technical possibilities to extend point-based CoPS towards imaging CoPS using camera detectors. This will involve a proof-of-principle study based on an emCCD camera to characterize defined samples in imaging mode as well as establishing a theoretical model for spatially-resolved photon statistic data evaluation. Furthermore, we want to evaluate the potential of alternative detection schemes such as APD arrays for imaging CoPS with higher time resolution.