August 2021 - René Platzer

Dr. René Platzer

MedUni Wien RESEARCHER OF THE MONTH August 2021

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

Selected Literature

1. Platzer R*, Rossboth BK*, Schneider MC, Sevcsik E, Baumgart F, Stockinger H, Schütz GJ, Huppa JB+, and Brameshuber M+. 2020. Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy. Nature Communications. 11:4993. *equal contribution, +corresponding author
2. Rossboth B, Arnold AM, Ta H, Platzer R, Kellner F, Huppa JB, Brameshuber M+, Baumgart F+, and Schütz GJ+. 2018. TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells. Nature Immunology. 19:821-827. +corresponding author
3. Platzer R and Huppa JB. T-cell Antigen Recognition – Lessons from the Past and Projections into the Future. 2020. Encyclopedia of Life Sciences (eLS). John Wiley & Sons, Ltd: Chichester. doi: 10.1002/9780470015902.a0001229.pub3.
4. Brameshuber, M, Kellner F*, Rossboth BK*, Ta H, Alge K, Sevcsik E, Göhring J, Axmann M, Baumgart F, Gascoigne NRJ, Davis SJ, Stockinger H, Schütz GJ, and Huppa JB+. 2018. Monomeric TCRs drive T cell antigen recognition. Nature Immunology. 19:487-496. *equal contribution, +corresponding author
5. Baumgart F, Arnold AM, Rossboth BK, Brameshuber M, and Schütz GJ+. 2018. What we talk about when we talk about nanoclusters. Methods and Applications in Fluorescence. 7:013001. +corresponding author