ROKE
• A high throughput 2P-FLIM microscope has been constructed.
• A new SFS instrument is under construction.
• Using the microscope we have taken SH images of several samples, testing water imaging, resonant SH fluorophore imaging and 2PF imaging. The images were compared in terms of quality to a modified version of an earlier microscope. Output: High throughput wide field SH imaging of giant unilamellar vesicles. M. Eremchev, D. Roesel, P.-M. Dansette, A. Michailovas, and S. Roke, Biointerphases 18, 2023; doi: 10.1116/6.0002640
• Measurements of water using high-throughput wide-field nonresonant second har-monic (SH) microscopy of membrane water shows that membrane potential fluctua-tions are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These tran-sient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. First test were also performed with living cells.
Output: 1 ) Passive transport of Ca2+ ions through lipid bilayers imaged by wide-field second harmonic microscopy, M. Eremchev, D. Roesel, C. S. Poojari, A. Roux, J. S. Hub and S. Roke, Biophys. J. (2023), 122(4):624-631
2) Ion induced transient potential fluctuations facilitate pore formation and cation transport through lipid membranes, D. Roesel, M. Eremchev, C. S. Poojari, J. S. Hub, and S. Roke, J. Am. Chem. Soc. (2022), 144, 51, 23352–23357.
ROUX
Over the last few months, students’ in-depth studies focussed on two critical systems for the project. They investigated gradients of flipper lifetime in cells and artificial lipid membranes, finding that these gradients correspond to membrane tension gradi-ents. Additionally, they developed an assay for oscillatory osmotic shocks, discover-ing that the coupling between volume and membrane tension weakens at high fre-quencies. Furthermore, they collected extensive data for a tutorial article on using flipper probes to measure membrane tension, which is currently under submission. A publication based on these findings is also in preparation. Output: 1) Garcia J-M., Guillamat P., Tomba C., Houzet L., Mehidi A., Aumeier C., Colom A., Roux A., Maintenance of membrane tension gradients in migrating and resting cells. Publication July 2024
2) Roffay, C., García-Arcos, J.M. Chapuis, P., López-Andarias, J., Schneider, F., Colom, A., Tomba, C., Meglio, I.D. Dunsig, V., Matile, S., et al. (2023). Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes. bioRxiv, 2022.2009.2028.509885.
