Accurate drift-invariant single-molecule force calibration using the Hadamard variance.

Area of Science:

• Biophysics
• Single-molecule biophysics
• Mechanobiology

Background:

• Single-molecule force spectroscopy (SMFS) is crucial for studying biological macromolecules.
• Multiplexed magnetic tweezers (MT) are ideal for probing forces below 1 pN, essential for analyzing noncovalent interactions.
• Accurate force calibration is critical for reliable measurements in MT experiments.

Purpose of the Study:

• To introduce and validate a novel Hadamard variance (HV) based method for calibrating magnetic tweezers (MT).
• To compare the performance of the HV method against established power spectral density (PSD) and Allan variance (AV) techniques.
• To assess the robustness of the HV method against common noise sources and experimental drift.

Main Methods:

• Development of a Hadamard variance (HV) analysis for MT force calibration.
• Simulations using bead-tether Brownian dynamics to mimic experimental MT systems.
• Comparative analysis of HV, PSD, and AV methods using simulated and experimental MT data, assessing performance under various noise and drift conditions.

Main Results:

• The HV method demonstrates comparable or superior precision and accuracy to PSD and AV methods.
• HV analysis yields lower force estimation errors across a range of signal-to-noise ratios (SNRs) and drift speeds.
• The HV method shows remarkable robustness against drift, maintaining consistent uncertainty levels.

Conclusions:

• The Hadamard variance (HV) method provides a robust approach for achieving sub-piconewton (pN) resolution and precision in multiplexed MT measurements.
• This method has the potential to advance the understanding of mechanosensitivity and force generation in biological systems.
• A Python implementation of the HV method is provided to enhance accessibility for the research community.