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TU Wien

3 ARTICLES PUBLISHED IN JoVE

Bioengineering

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Markus Axmann¹, Gerhard J. Schütz¹, Johannes B. Huppa²

¹Institute for Applied Physics - Biophysics, Vienna University of Technology, ²Institute for Hygiene and Applied Immunology, Medical University of Vienna

This manuscript describes how to conduct (single molecule) Förster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Bioengineering

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Markus Axmann¹, Gerhard J. Schütz¹, Johannes B. Huppa²

¹Institute of Applied Physics - Biophysics, Vienna University of Technology, ²Institute for Hygiene and Applied Immunology, Immune Recognition Unit, Medical University of Vienna

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bioengineering

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Lukas Schrangl¹, Janett Göhring², Florian Kellner², Johannes B. Huppa², Gerhard J. Schütz¹

¹Institute of Applied Physics, TU Wien, ²Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna

This article presents a method for spatiotemporal analysis of mobile, single-molecule Förster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.