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

• Applicant: Dr. Daniela Täuber
• Subject Area: Biophysics
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 439139881

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.

• DFG Programme: Research Grants