Live Cell 2D polarization imaging (Live2DPOLIM) – an imaging technique for observation of nanoscale protein aggregation and molecular (re-)arrangements in live cells

拟议的Live2DPOLIM旨在建立最近发展的二维偏振荧光成像（2D POLIM），用于生物医学研究，包括免疫反应，靶向药物释放和全身感染引起的器官衰竭等高兴趣领域的活细胞研究。2d偏振分辨测量与建模相结合的定量分析将使研究活细胞中蛋白质的纳米级聚集和分子细胞结构的重新排列独立于体外和体内的分子排列成为可能，这是迄今为止无法通过现有技术进行评估的，因此有助于对感染相关损伤、免疫反应和治疗的基本理解。监测纳米级结构排列的能力是基于类似荧光标记（homo-FRET, emFRET）之间Förster共振能量转移的评估，这是一种成熟的2至10纳米范围内的纳米统治者，这一空间尺度仍然难以用传统甚至超分辨率显微镜获得。Live2DPOLIM与基于红外激发和原子力显微镜（IR-AFM）检测的新兴无标记方法相结合，补充了细胞结构的纳米级聚集信息和细胞表面的高分辨率化学信息。这将提供与所研究细胞的应用处理相关的细胞膜上的分子重排，并允许评估应用于细胞结构的染色方法的性能，从而验证与Live2DPOLIM定量分析使用的建模的准确性。Live2DPOLIM的强大之处在于它能识别细胞结构的纳米聚集。然而，由于需要获得高质量的偏振特性，纳米聚集体的定位受传统光学分辨率的制约。因此，我们建议实现偏振分辨单分子定位显微镜（SMLM）的附加分析代码，通过将离体和活细胞中染色结构成像的光学分辨率提高到约50 nm，补充了Live2DPOLIM的高分辨局部信息和IR-AFM的化学信息。这种结合将使使用Live2DPOLIM观察到的纳米聚集能够分配到偏振分辨SMLM提供的超分辨率图像中的特定细胞结构特征，这将进一步提高对感染相关损伤和治疗的理解。

The suggested Live2DPOLIM aims at establishing the recently developed two-dimensional polarization fluorescence imaging (2D POLIM) for biomedical research including live cell investigation in the high interest areas of immune response, targeted drug release, and organ failure caused by systemic infection. Quantitative analysis of 2D-polarization resolved measurements combined with modeling will enable the investigation of nanoscale aggregation of proteins and re-arrangements of molecular cellular structures in live cells ex vivo and in vivo independent of molecular alignment which so far cannot be assessed by established techniques, and thus contribute to the fundamental understanding of infection-related damage, immune response, and therapy. The power to monitor nanoscale structural arrangements is based on the evaluation of Förster resonance energy transfer between similar fluorescence labels (homo-FRET, emFRET), which is a well-established nanoruler in the range of 2 to 10 nm, a spatial scale that still is hard to access with conventional and even superresolution microscopy.Combining Live2DPOLIM with emerging label-free methods based on infrared excitation with detection using atomic force microscopy (IR-AFM) complements the information on nanoscale aggregation of cellular structures with highly resolved chemical information of cell surfaces. This will provide access to molecular re-arrangement on cell membranes associated with the applied treatments of the investigated cells, and it allows for evaluating the performance of staining methods applied to cellular structures, thereby, validating the accuracy of modeling used with quantitative analysis of Live2DPOLIM.The power of Live2DPOLIM is the discrimination of nano-aggregation of cellular structures. Yet, the localization of the nano-aggregates is governed by conventional optical resolution due to the requirement to retrieve high quality polarization properties. Thus, the implementation of additional analysis code for polarization resolved Single Molecule Localization Microscopy (SMLM) is suggested, which complements the highly resolved local information using Live2DPOLIM and chemical information using IR-AFM by enhancing the optical resolution to about 50 nm for imaging stained structures ex vivo and in live cells. This combination will enable the assignment of the nano-aggregation observed using Live2DPOLIM to specific cellular structural features in the superresolution images provided from application of polarization resolved SMLM, which will further improve the understanding of infection related damage and therapy.