CALISTO: A Standardized and Open-Source Software for Magnetic Tweezer Force Calibration

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.