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

Live2DPOLIM旨在建立最近发展起来的二维偏振荧光成像(2D POLIM)，用于生物医学研究，包括在免疫反应、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极化分辨测量的定量分析与建模相结合，将使我们能够研究活细胞内蛋白质的纳米级聚集和分子细胞结构的重排，而不依赖于迄今无法用现有技术评估的分子排列，从而有助于从根本上理解感染相关的损伤、免疫反应和治疗。监测纳米尺度结构排列的能力是基于相似荧光标记(HOMO-FRET，emFRET)之间的Förster共振能量转移的评估，这是一个在2到10 nm范围内公认的纳米尺度，传统甚至超分辨率显微镜仍然很难获得这个空间尺度。Live2DPOLIM与新兴的基于红外激发和原子力显微镜(IR-AFM)检测的无标记方法相结合，补充了细胞结构纳米尺度聚集的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的途径，并允许评估应用于细胞结构的染色方法的性能，从而验证用于Live2DPOLIM定量分析的建模的准确性。Live2DPOLIM的能力是对细胞结构的纳米聚集的区分。然而，由于需要恢复高质量的偏振特性，纳米聚集体的局域化受传统光学分辨率的制约。因此，建议实现偏振分辨单分子定位显微镜(SMLM)的附加分析程序，该程序通过将光学分辨率提高到约50 nm来补充Live2DPOLIM的高分辨局部信息和IR-AFM的化学信息，用于在体外和活细胞中成像染色结构。这种结合将使使用Live2DPOLIM观察到的纳米聚集能够在应用偏振分辨SMLM提供的超分辨率图像中指定特定的细胞结构特征，这将进一步提高对感染相关损害和治疗的理解。

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.