Biophysical Society 63rd Annual Meeting | Program Guide

Room 303: Monday, March 4 9:30 AM – 11:00 AM Bruker Corporation Advances in Dye Development and Microscopy for Live Cell Superresolution Microscopy with the Vutara 352 Expanding the frontier of super-resolution imaging requires advances in both microscopy hardware and fluorescent labels. Here we describe a cooperative effort to improve both technological fronts with the ultimate goal of live-cell super-resolution microscopy. Bruker’s Vutara 352 super-resolution microscope has been designed for live-cell super- resolution microscopy with both high spatial and temporal resolution capabilities. The patented biplane module allows simultaneous two- color imaging in 3D while the sCMOS detector enables fast imaging of biological phenomena. Although this microscope system is capable of live-cell super-resolution imaging, it has been stymied by limitations in the current generation of live-cell-compatible fluorophores. Extant live- cell probes are either fluorescent proteins with low photon counts—and therefore low localization precision—or organic dyes, which require high laser power resulting in phototoxicity in living samples. To remedy this problem, we developed spontaneously blinking (SB) versions of the Janelia Fluor and Alexa Fluor dyes, which blink under physiological conditions at low laser power while still providing high photon counts. In particular, the spontaneously blinking Janelia Fluor 549 (SB-JF549) and red-shifted SB-JF646 are cell-permeable and are easily conjugated to HaloTag or SNAP-tag ligands, making them ready to use in live cell multi- color superresolution experiments. The SB dyes, in combination with the Vutara 352, provide a powerful methodology for simultaneous imaging, localization and visualization of live-cell single-molecule localization data, while offering numerous statistical tools to quantify the data into publishable results. Speaker Robert Hobson, Applications Scientist, Bruker Corporation

11:30 AM – 1:00 PM Asylum Research Capturing Biochemical Reactions with Video-Rate AFM Oxford Instruments Asylum Research will present the latest data acquired with its Cypher VRS, the world’s first and only full-featured video-rate AFM. The Cypher VRS Atomic Force Microscope sets a new standard with easy operation—enabling high resolution imaging of dynamic events at high speeds, up to 625 lines/second which corre- sponds to about 10 frames per second. This speed is about 300x faster than typical AFMs and 10x faster than current “fast scanning” AFMs. One of the strengths of traditional AFMs is its capability to monitor dynamic events in near-native conditions (i.e. in liquid at biologically relevant temperatures). However, capturing biological processes in real- time has been challenging up until now. Video rate AFMs provide that temporal resolution, allowing researchers to observe the progression of these reactions and capture kinetics. Video rate AFMs have allowed researchers to conduct a new set of experiments including biochemical reactions, membrane dynamics, conformational changes, self-assembly and degradation. In most cases, the spatial resolution is not compro- mised enabling researchers to locate the target or active site while tracking the progression of the reaction. They can observe structural dynamics of biomolecules and then correlate it to their function. We will present a set of data to illustrate the potential of this new capa- bility. Examples include DNA digestion and cleavage, DNA origami con- formation changes, protein fiber assembly, membrane dynamics includ- ing molecular structure and rearrangement in the bacteriorhodopsin membrane, lipid bilayer growth, assembly of Type I collagen into fibrils and dynamic motion of CTAB hemi-micelles at the solid (HOPG) – liquid (aqueous buffer) interface. Speaker Sophia Hohlbauch, Applications Scientist, Asylum Research

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