Biology subjects

Cnossen, J.

Publications and source records attributed to Cnossen, J..

6 recordsLinked to original sources

bioRxiv2026

A multimodal, correlative magnetic tweezers-TIRF platform for high-throughput single-molecule interrogations

Single-molecule techniques can resolve biological reactions at unmatched detail, but their low throughput and single-modality readouts have kept them out of data-intensive pipelines such as omics and drug discovery, and beyond reach of low-yield biological systems. Here we introduce a multimodal platform integrating high-throughput magnetic tweezers with ultra-wide-and flat-field objective-based total internal reflection fluorescence, enabling simultaneous force, torque, multicolor fluorescence, and temperature-dependent measurements on up to thousands of individual molecules in parallel and in real time. We demonstrate accurate single-molecule Forster resonance energy transfer (smFRET) for prism-based spectral imaging, capture temperature-dependent hairpin folding dynamics at high temporal resolution with smFRET and use correlative torque-fluorescence measurements to unravel the open-complex formation dynamics during bacterial transcription initiation. By unifying high resolution, throughput, and multimodal readout, this platform enables multidimensional dissection of complex biomolecular reactions with high statistical confidence, unlocking single-molecule biophysics for integration with drug discovery, omics, and cryo-EM workflows.

biophysics↗

bioRxiv2025

Patterned illumination enables denser deep-learning based single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) improves resolution beyond diffraction limits by imaging cellular structures at the nanometer scale. Recent advances include using modulation patterns to improve localization precision, and deep learning to accurately process high-density samples with overlapping fluorophore emissions, thus improving imaging speed. A method combining these two advances, SIMCODE, is presented here, allowing high-density modulation-enhanced SMLM. SIMCODE achieved resolution improvements in high-density areas compared to SMLM, deep learning-based SMLM (DECODE), and modulation-enhanced SMLM alone (SIMFLUX). In DNA-PAINT imaging of COS-7 cells, SIMCODE showed improvements in the Fourier Ring Correlation and resolution-scaled Pearson coefficient, with overall improvement increasing as imaging buffer concentration increased five-fold. Modulation-enhanced localization microscopy combined with deep learning thus produced higher quality reconstructions at higher emitter densities (i.e., [~]3x the number of detected spots). This will enable faster imaging, higher labeling densities, and more flexibility in fluorophore choice, which are important for studying dynamic processes and densely labeled structures.

biophysics↗

bioRxiv2024

Using splines for point spread function calibration atnon-uniform depths in localization microscopy

Single-molecule localization microscopy methods extensively leverage the microscope point spread function (PSF) for fitting the molecules. Calibrating an accurate PSF model is especially difficult in the presence of depth-dependent aberrations which alter the PSF shape depending on the imaging depth. The aberrations at depths of a few micrometers become substantial enough to considerably impoverish the conventional calibration methods performance. In our work, we propose a novel spline model which enables the depth-dependent PSF model calibration by interpolating between the beads at arbitrary depths. We show that diffspline reduces the PSF intensity overestimation by 67.8 percentage points and underestimation by 21.8 percentage points. Moreover, it eliminates the depth-dependent bias and improves the localization precision two-fold compared to previous approaches.

biophysics↗

bioRxiv2021

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor{sigma} to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPO is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with{sigma} and the RNAP {beta} subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

biophysics↗

bioRxiv2021

Drift correction in localization microscopy using entropy minimization

Localization microscopy offers resolutions down to a single nanometer, but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal precision and bias, which leaves room for improvement. Here, we minimize a bound on the entropy of the obtained localizations to efficiently compute a precise drift estimate. Within practical compute-time constraints, simulations show a 5x improvement in drift estimation precision over the widely used RCC algorithm. The algorithm operates directly on fluorophore localizations and is tested on simulated and experimental datasets in 2D and 3D. An open source implementation is provided, implemented in Python and C++, and can utilize a GPU if available.

biophysics↗

bioRxiv2019

Localization microscopy at doubled precision with patterned illumination

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field-of-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.

molecular biology↗