Biophysical Society 65th Annual Meeting Program Guide

curve which provides insights into the underlying dynamics as well as the concentration of the observed species [2]. Until now, the concentra- tion 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 fluo- rophore 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 concen- trations 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 implementa- tion [3]. By measuring the diffusion of fluorophores in solution and cyto- plasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes on a turn-key instrument. This may reduce the bias when performing live cell measure- ments where varying expression levels occur routinely and increases the experimental flexibility. In STED-FCS data quality suffers from the fluores- cence 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. J. Lackowicz, Principles of Fluorescence Spectroscopy, Third. Boston, MA: Springer US, 2006. 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 Break 1:30 pm - 2:00 pm Poster Presentations and Late Posters 2:00 pm - 3:30 pm Exhibitor Presentation LUMICKS 3:30 pm - 4:00 pm 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 ob- serve a biological process frommultiple points of view. Using LUMICKS’ groundbreaking C-Trap ® Optical Tweezers – Fluorescence & Label-free Microscopy, you can simultaneously visualize individual molecules 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. [2] [3] Giulia Ossato, Product Manager, Leica Microsystems Julia Roberti, Product Manager, Leica Microsystems Falk Schneider, University of Oxford [1]

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 polypep- tide 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 translo- cation 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 Peer-to Peer Networking Mixer 3:30 pm - 5:30 pm Looking for an opportunity to connect with your peers? This event is a great chance to compare notes with colleagues, make new connections, and dis- cuss one-on-one your unique solutions to issues that arise in your particular career stage. Virtual networking space will be provided for Annual Meeting attendees at all career levels, from undergrads to established researchers. Exhibitor Presentation Carl Zeiss Microscopy LLC 4:00 pm - 4:30 pm Discovering the Subcellular Dynamics of Life with ZEISS Lattice Lightsheet 7 In order to best understand the world around us it is necessary to observe microscopic specimins in as natural a state as possible. This requires a transition from imaging fixed to live specimins and expanding from 2D to 3D model organisms. The drive towards live-cell imaging over long timeframes and at high volume speeds brings new challenges. There is evidence that traditional imaging techniques can influence the behaviour of specimins due to phototoxicity, thus affecting the integrity of the results. The most influential technological breakthroughs which address these chal- lenges have been modifications to the shape of the excitation light. Classical laser-based imaging approaches utilize a gaussian excitation beam which is focussed to a spot or a sheet and scanned as required to excite the sample. As an alternative approach, bessel beams have been combined to introduce a structured pattern to the beam profile. The resulting ‘lattice’ of light has many benefits for live imaging. The most notable are a reduction of light ex- posure due to significant improvement in signal to noise while maintaining high resolution and optical sectioning. With lattice-lightsheet microscopy it is possible to capture dynamics at previously unreachable combinations of acquisition speed and resolution over hours and even days. This talk will describe how the ZEISS Lattice Lighsheet 7 makes long-term volumetric imaging of living cells with subcellular resolution possible without having to change your standard sample preparation protocols to accomdate the instrument. With automatic alignment and easy acquisi- tion workflows, lattice light-sheet imaging is now as accessible as using a standard inverted microscope. Join us for this webinar to learn how ZEISS Lattice Lightsheet 7 allows you to discover the subcellular dynamics of life. Speaker Renée Dalrymple, Product Marketing Manager – Life Sciences Lattice Line, Carl Zeiss Microscopy LLC Biophysical Society Business Meeting 4:00 pm - 4:30 pm

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