Biophysical Society 65th Annual Meeting Program Guide

1:30 PM – 2:00 PM Leica Microsystems High Photon-Count Rate FCS and STED-FCS to Study Diffu- sion Dynamics in Live Cells Investigating diffusion dynamics of proteins and small molecules has become a routine measurement all across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligo- merization, molecular interactions or cellular (membrane) heteroge- neities [1]. Fluorescence correlation spectroscopy (FCS) is a versatile tool to determine diffusion dynamics in membranes (2D diffusion) and solution or the cytoplasm (3D diffusion). Measuring the intensity fluctuations over time due to the diffusion of molecules through the observation volume is the basis for FCS. Temporal autocorrelation of the signal allows for the calculation of the autocorrelation curve which provides insights into the underlying dynamics as well as the concentration of the observed species [2]. Until now, the concentration regime for reliable measurements has been limited by the detection electronics which could not efficiently and accurately time-tag photons at high photon-count rates. This restricted the range of measurable fluorophore concentrations and data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED)-FCS. In this talk, we will show the applicability and reliability of FCS at high photon-count rates (average intensities of more than 1 MHz and concentrations higher than 1 µM) using novel detection equipment based on hybrid detectors, namely HyD SMDs, and real-time gigahertz sampling of the photon stream using the Leica SP8 STED FALCON FCS implementation [3]. By measuring the diffusion of fluorophores in solu- tion and cytoplasm of live cells, as well as in model and cellular mem- branes, we show that accurate diffusion and concentration measure- ments are possible in these previously inaccessible high photon count regimes on a turn-key instrument. This may reduce the bias when performing live cell measurements where varying expression levels occur routinely and increases the experimental flexibility. In STED-FCS data quality suffers from the fluorescence depletion and can be greatly improved by using higher confocal count rates. The presented data show a path towards robust FCS and STED-FCS measurements in living cells. Speakers E. Sezgin et al., “Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED–FCS,” Nat. Protoc., vol. 14, no. 4, pp. 1054–1083, Apr. 2019. [2] J. Lackowicz, Principles of Fluorescence Spectroscopy, Third. Boston, MA: Springer US, 2006. [3] F.Schneider et al., “High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy,” J. Phys D: Appl. Phys., vol. 53, no. 16, 2020 Giulia Ossato, Product Manager, Leica Microsystems Julia Roberti, Product Manager, Leica Microsystems Falk Schneider, University of Oxford [1]

3:30 PM – 4:00 PM LUMICKS

Correlative Force–Fluorescence Measurements to Reveal the Dynamic Life of Single Biomolecules: Latest Technology Advancements by LUMICKS, and Latest Findings on Protein Disaggregation by Professor Sander Tans To decipher complex molecular interactions, you need to be able to observe a biological process from multiple points of view. Using LUMICKS’ groundbreaking C-Trap® Optical Tweezers – Fluorescence & Label-free Microscopy, you can simultaneously visualize individual mol- ecules in real time and measure dynamic biological processes in great detail. During our webinar, we will reveal how our latest technology develop- ment, the Trap Distance Lock, allows you to measure biomolecular equilibrium dynamics with unprecedented stability over extremely long periods of time. This new feature offers the ultimate system stability that enables you to capture the rarest, fastest, and smallest conforma- tional changes that underlie the energy landscapes of biomolecules. After a brief introduction by LUMICKS, we are honored to give the floor to our invited speaker Prof. Sander Tans who will present his work on polypeptide loop extrusion using correlative force–fluorescence measurements. Re-dissolving protein aggregates is crucial to cells, but the molecular basis has remained unknown. Using combined optical tweezers and single-molecule fluorescence detection, Prof. Tans and his team showed that the disaggregase ClpB extrudes loops of protein chains through its central pore, and hence forcibly extracts protein chains from aggregates. The data reveal notable processivity, power, step-dynamics, and switching between translocation modes. Protein disaggregation can thus be highly deterministic and energy-driven process, while polypeptide loop extrusion may be exploited by other systems including p97/cdc48. Speakers Olivier Heyning, CEO and Founder, LUMICKS Aida Llauró Portell, Senior Application Scientist, LUMICKS Sander Tans, AMOLF and Delft University of Technology, The Netherlands

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