Developing a new biophysical tool to combine magneto-optical tweezers with super-resolution fluorescence microscopy

TL;DR

This paper introduces a combined biophysical tool integrating magnetic and optical tweezers with super-resolution fluorescence microscopy, enabling detailed, simultaneous mechanical manipulation and imaging of single molecules for advanced molecular studies.

Contribution

The work develops a novel, reproducible system that merges tweezing and super-resolution imaging, allowing direct observation of molecular transformations and protein binding during mechanical assays.

Findings

Achieved ~30 nm localization precision of fluorescent dyes on DNA

Enabled simultaneous force measurement and super-resolution imaging

Facilitated direct visualization of molecular topological changes

Abstract

We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM) achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing a system which combines tweezing and super-resolution fluorescence imaging. The instrument also features an acousto-optic deflector that temporally divides the laser beam to form multiple traps for high throughput statistics collection. Our motivation for developing the new tool is to enable direct observation of detailed molecular topological transformation and protein binding event localisation in a stretching/twisting mechanical assay that previously could hitherto only be deduced indirectly from the end-to-end length variation of DNA. Our approach is simple and robust enough for reproduction in the lab without the requirement of precise hardware engineering, yet is capable of unveiling the elastic and dynamic properties of filamentous molecules that have been hidden using traditional tools.