Live Cell 2D polarization imaging (Live2DPOLIM) – an imaging technique for observation of nanoscale protein aggregation and molecular (re-)arrangements in live cells
Final Report Abstract
Advanced polarization-resolved fluorescence microscopy provides complementary information on macromolecular organisation in cells, contributing to our understanding of cell functionality and pathologic pathways. We have developed a speeded-up setup for 2-dimensional polarization-resolved fluorescence imaging (2DPOLIM) by implementing polarization modulation using a liquid crystal polarization rotator (LCR) in the excitation together with a 4-channel detection. 2DPOLIM complements single molecule localization microscopy (SMLM) by providing up to mm-sized overviews on average polarization parameters and nanoscale macromolecular organization. Our results from application of 2DPOLIM to hepatocytes, fibroblasts10 and single F-Actin fibrils stained by a Phalloidin-dye complex confirm the applicability of 2DPOLIM for evaluating variations in F-actin organization. Due to the impact of the pandemic, in particular, live cell imaging has been delayed and is expected within the upcoming year from our planned work on using and improving 2DPOLIM. For the evaluation of results obtained from Phalloidin-dye labelled F-Actin using 2DPOLIM, we developed an ex silico model for single F-Actin fibrils based on a model for co-expressed green fluorescent protein (GFP) in a mouse model. Dye molecules linked to Phalloidin may perform a statistical motion of the chromophore causing orientational fluctuations of the emission. To understand its influence on experimental results, a molecular dynamics (MD) simulation of three such complexes in F-Actin was developed. We additionally implemented fluorescence lifetime imaging microscopy (FLIM) for investigating macromolecular organization in our lab. First results applying FLIM to Phalloidin-dye stained cancer cells showed increased fluorescence lifetimes for cortical F-Actin compared to those obtained for cytoskeletal Actin filaments. Due to these promising results, a more extensive study, including also fluorescence labels suitable for life cell imaging has been started together with our collaborators at the UKJ. As suggested in our project proposal, we complemented our research on macromolecular organization by using emerging high-resolution nanoscale infrared (IR) spectroscopic imaging techniques which combine powerful IR illumination with mechanical detection by atomic force microscopy (IR-AFM). We demonstrated sub-molecular spectral resolution of F-actin with unprecedented spatial resolution in hyperspectral data obtained using mid-IR photoinduced force microscopy (IR-PiFM or PiF-IR). Further results using PiF-IR on macromolecules, cells and cell organelles have been obtained. IR-AFM methods exert orientation-sensitive detection, which was used for structural evaluation in a self-organized heterodyne polymer film. To meet the unprecedented spatial resolution of PiF-IR a quantumchemical calculation of polarization-resolved IR spectra of a di-peptide has been performed.
Publications
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Raw data for Joseph et al.15
Täuber, Daniela
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„hyPIRana“ homebuilt PiF-IR hyperspectral analysis software used for data analysis in Joseph et al.15
S. Unger, M. Ali, M. Soltaninezhad, R. Lachmann, R. Heintzmann & D. Täuber
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Imaging Aggregation of Histologically Stained F-Actin Ex Vivo Using the Contrast in Förster Resonance Energy Transfer Obtained from 2D Polarization Fluorescence Imaging (2D-POLIM). In European Light Microscopy Initiative 2021; Royal Microscopical Society, 2021
D. Täuber, J. Shi, P. Babic, K.-J. Benecke, M. Bauer, R. Heintzmann, I. G. Scheblykin & A. T. Press
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Intrinsic polarization-sensitive organic photodetector with self-assembled all-polymer heterojunction. Applied Physics Letters, 121(23).
Luo, Xuhao; Xue, Yingying; Wu, Juntao; Cai, Wanzhu; Täuber, Daniela; Malovicho, Ivan; Sava, Bogdan; Cen, Guobiao; Lu, Xing; Zhao, Chuanxi; Scheblykin, Ivan G.; Yu, Jianhui; Mai, Wenjie; Liu, Feng; Wang, Ergang & Hou, Lintao
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Topological Artifacts in Mid-IR Photo-Induced Force Microscopy (PiF-IR); Regensburg, 2022
S. Barua, H. Gadher, U. Hübner & D. Täuber
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Fast Excitation Modulation via Liquid Crystal Polarization Rotator in 2D Polarization Fluorescence Imaging (2D POLIM) and Compensation of Depolarization Caused by Dichroic Mirrors; Klosters, Switzerland, 2023
Y. Wang, A. Hafeez, M. Soltaninezhad, R. Heintzmann & D. Täuber
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High resolution chemical imaging of surfaces using infrared spectroscopic photo-induced force microscopy (PiF- IR) and its requirements for data analysis., WG1 workshop, E-Cost Action Ca21111 OneHealthdrugs, Oct. 2023 online
M. Ali, R. Schneider, S. Unger, U. Neugebauer, R. Heintzmann & D. Täuber
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Nanochemische Bildgebung in der Infektionsforschung, Science Slam, Set up : Jena | Gründungs- und Innovationstag 26.11.23, Nucleus Jena
Täuber, Daniela
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F-Actin Fluorescence Staining Evaluated Using 2D Polarization Fluorescence Imaging (2DPOLIM), Structured Illumination Microscopy (SIM) and Fluorescence Lifetime Imaging (FLIM); Klosters, Switzerland, 2024
S. Cheng, S. Gjeci, A. Rusevski, P. Then, E. Sunil, S. Adak, H.-D. Arndt, R. Heintzmann, A. T. Press & D. Täuber
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Nanoscale chemical characterization of secondary protein structure of F-Actin using mid-infrared photoinduced force microscopy (PiF-IR). Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 306, 123612.
Joseph, Jesvin; Spantzel, Lukas; Ali, Maryam; Moonnukandathil, Joseph Dijo; Unger, Sebastian; Reglinski, Katharina; Krafft, Christoph; Müller, Anne-Dorothea; Eggeling, Christian; Heintzmann, Rainer; Börsch, Michael; Press, Adrian T. & Täuber, Daniela
