Year: 2026
Type: preprint
Abstract: ABSTRACT Magnetic tweezers (MT) enable multiplexed single-molecule force spectroscopy while applying controlled forces and torques to biomolecules. They resolve forces below 1 pN and maintain constant forces over extended periods, but accurate calibration remains challenging due to drift, tracking artifacts, camera noise, and bead variability. Here, we present CALISTO (CALIbration Suite for magnetic Tweezer Operation), an open-source software for MT force calibration. CALISTO analyzes position traces of DNA-tethered magnetic beads and surface-bound reference beads for drift correction. It implements three thermal-motion-based calibration methods: power spectral density, Allan variance, and Hadamard variance, with the latter providing increased robustness against drift and correlated measurement noise. The graphical user interface combines automated analysis with user-guided quality control, including reference bead selection, z-offset determination, magnet-position classification, bead-wise force estimation, and tether extension analysis. Together, these steps generate a master force calibration curve. We systematically evaluated their effects on calibration performance. The largest improvement resulted from automated filtering of problematic force plateaus using time-series characterization to identify traces affected by drift, tracking artifacts, or irregular fluctuations. Filtering reduced scatter and improved force estimation while preserving the underlying force–magnet position relationship. CALISTO thus provides a standardized, reproducible framework for reliable magnetic-tweezers force calibration. STATEMENT OF SIGNIFICANE Magnetic tweezers enable highly sensitive and long-duration measurements of forces and torques in single biomolecules, but their accuracy critically depends on reliable force calibration. CALISTO provides an open-source, standardized framework that integrates force calibration with automated quality control and quantitative assessment of experimental data. By identifying problematic measurements caused by drift, tracking artifacts, or irregular bead fluctuations, CALISTO substantially improves calibration reliability while preserving the underlying force–position relationship. The software implements complementary thermal-motion-based calibration methods and combines automated processing with user-guided validation in an accessible graphical interface. CALISTO facilitates reproducible magnetic-tweezers experiments across different experimental conditions and provides a practical foundation for more reliable quantitative single-molecule force measurements.
Institutions: University of Augsburg, Utrecht University
Language: English
Subfield: Atomic and Molecular Physics, and Optics
Field: Physics and Astronomy
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7222974361