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

建议Live2DPOLIM的目的是建立最近开发的二维偏振荧光成像（2D POLIM）的生物医学研究，包括在免疫反应，靶向药物释放，以及全身感染引起的器官衰竭的高兴趣领域的活细胞调查。2D偏振分辨测量的定量分析与建模相结合，将使研究的纳米级聚集的蛋白质和重新安排的分子细胞结构在活细胞中的离体和在体内的分子排列，到目前为止，不能通过建立的技术进行评估，从而有助于感染相关的损伤，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对相似荧光标记之间Förster共振能量转移的评估（homo-FRET，emFRET），其是在2至10 nm范围内的公认的纳米粒子，一个空间尺度，仍然是难以访问的传统，甚至超分辨率显微镜。结合Live2DPOLIM与新兴的标签-基于红外激发并使用原子力显微镜（IR-AFM）检测的自由方法用细胞表面的高分辨化学信息补充了关于细胞结构的纳米级聚集的信息。这将提供与所研究细胞的应用治疗相关的细胞膜上的分子重排的途径，并且它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定量分析一起使用的建模的准确性。Live2DPOLIM的能力是区分细胞结构的纳米聚集。然而，由于需要检索高质量的偏振特性，纳米聚集体的定位由常规的光学分辨率决定。因此，实施偏振分辨单分子定位显微镜（SMLM）的额外的分析代码的建议，它补充了高分辨率的本地信息使用Live2DPOLIM和化学信息使用IR-AFM通过提高光学分辨率到约50 nm的离体和活细胞中的染色结构的成像。这种组合将使得能够将使用Live2DPOLIM观察到的纳米聚集分配给由偏振分辨SMLM的应用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.