Numerous biophysical techniques such as magnetic tweezers, flow stretching assays, or tethered particle motion assays rely on the tracking of spherical beads to obtain quantitative information about the individual biomolecules to which these beads are bound. The determination of these beads' coordinates from video-based images typically forms an essential component of these techniques. Recent advances in camera technology permit the simultaneous imaging of many beads, greatly increasing the information that can be captured in a single experiment. However, computational aspects such as frame capture rates or tracking algorithms often limit the rapid determination of such beads' coordinates. Here, we present a scalable and open source software framework to accelerate bead localization calculations based on the CUDA parallel computing framework. Within this framework, we implement the Quadrant Interpolation algorithm in order to accurately and simultaneously track hundreds of beads in real time using consumer hardware. In doing so, we show that the scatter derived from the bead tracking algorithms remains close to the theoretical optimum defined by the Cramer-Rao Lower Bound. We also explore the trade-offs between processing speed, size of the region-of-interests utilized, and tracking bias, highlighting in passing a bias in tracking along the optical axis that has previously gone unreported. To demonstrate the practical application of this software, we demonstrate how its implementation on magnetic tweezers can accurately track (with ∼1 nm standard deviation) 228 DNA-tethered beads at 58 Hz. These advances will facilitate the development and use of high-throughput single-molecule approaches.

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in fieldof-view. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures and tubulin in cells, using DNA-PAINT and STORM imaging. Single-molecule localization microscopy 1,2,3 circumvents the diffraction limit using centroid estimation of sparsely activated, stochastically switching, single-molecule fluorescence images. Improvement over state-of-the-art image resolutions of around 20 nm towards values below 5 nm is desired for truly imaging at the molecular scale. This needs improvements in labelling strategy to reduce linker sizes 4,5,6,7 and methods to overcome low labelling density such as data fusion 8 , but also a step in localization precision. Efforts so far have targeted an increase in the number of detected photons N by chemical engineering of brighter fluorophores 9 , or by avoiding photo-bleaching via e.g. cryogenic techniques 10,11,12. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection.

To study the behavior of biological macromolecules and enzymatic reactions under force, advances in single-molecule force spectroscopy have proven instrumental. Magnetic tweezers form one of the most powerful of these techniques, due to their overall simplicity, non-invasive character, potential for high throughput measurements, and large force range. Drawbacks of magnetic tweezers, however, are that accurate determination of the applied forces can be challenging for short biomolecules at high forces and very time-consuming for long tethers at low forces below ∼1 piconewton. Here, we address these drawbacks by presenting a calibration standard for magnetic tweezers consisting of measured forces for four magnet configurations. Each such configuration is calibrated for two commonly employed commercially available magnetic microspheres. We calculate forces in both time and spectral domains by analyzing bead fluctuations. The resulting calibration curves, validated through the use of different algorithms that yield close agreement in their determination of the applied forces, span a range from 100 piconewtons down to tens of femtonewtons. These generalized force calibrations will serve as a convenient resource for magnetic tweezers users and diminish variations between different experimental configurations or laboratories.

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny fieldof-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures. Main textSingle-molecule localization microscopy 1,2,3 circumvents the diffraction limit using centroid estimation of sparsely activated, stochastically switching, single-molecule fluorescence images. Improvement over state-of-the-art image resolutions of around 20 nm towards values below 5 nm is desired for truly imaging at the molecular scale. This needs improvements in labelling strategy to reduce linker sizes 4,5,6,7 and methods to overcome low labelling density such as data fusion 8 , but also a step in localization precision. Efforts so far have targeted an increase in the number of detected photons by chemical engineering of brighter fluorophores 9 , or by avoiding photo-bleaching via e.g. cryogenic techniques 10,11,12 . These improvements scale the localization precision with ( % ) ⁄ , with the fluorescence emission wavelength, and the microscope objective numerical aperture 13 .Recently, a new concept called MINFLUX was proposed 14 , in which a doughnut illumination spot is shifted over an area of size ~50 nm, and the position of a single molecule in the scan range is determined by triangulation based on the detected photon count for the different doughnut positions.