Developing a New Biophysical Tool to Combine Magneto-Optical Tweezers with Super-Resolution Fluorescence Microscopy

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.

Single-molecule techniques in biophysics: a review of the progress in methods and applications

“…Helmholtz coils are the simplest geometry to achieve a near-uniform magnetic field. Two pairs arranged perpendicular to each other can rotate the bead along one axis [277][278][279][280] whereas three pairs can rotate along all three directions [281]. Although one pair cannot rotate the bead by itself, it has found application in oscillating the beads [75].…”

Single-molecule fluorescence microscopy review: shedding new light on old problems

“…Here, the researchers used the native photoblinking behaviour of the common metabolite FAD inside a binding site of the enzyme cholersterol oxidase to demonstrate that its activity could be affected by a type of ‘molecular memory’ stored in the molecular confirmation [ 16 ]. Single-molecule fluorescence microscopy approaches since then have uncovered many fundamental molecular scale biological processes that were previously not studied primarily due to the limitations imposed by population methods, including studies of the bacterial flagellar motor rotation [ 17 – 21 ], protein folding, translocation and movement [ 11 , 22 – 25 ], signal transduction [ 26 ], biopolymer mechanics [ 27 – 32 ], DNA replication and remodelling [ 33 – 37 ], oxidative phosphorylation [ 38 – 41 ], as well as biomedically relevant areas such as the probing of processes relating to infection and general pathology [ 42 – 44 ], cell division mechanisms [ 45 ], mitochondrial protein dynamics [ 46 ], viral infection processes [ 47 ], endocytocis and exocytosis pathways [ 48 ], osmolarity receptor dynamics [ 49 ], cell wall synthesis [ 50 ], and structural dynamics of DNA [ 51 ]. This list above should not be taken as exclusive nor exhaustive, but rather we present it here to exemplify the very wide range of biological processes to which single-molecule fluorescence microscopy tools have been applied.…”

Optical Imaging of Magnetic Particle Cluster Oscillation and Rotation in Glycerol

“…Two sets of wire spools in a quadrupole configuration can be used to control magnetic particle cluster oscillation and rotation [ 26 ]. To image the particle cluster motion with an optical microscope, a microscope insert was engineered to fit within the stage of Lecia’s DMI6000 and DMI3000 inverted microscopes (Leica Microsystems Inc., Buffalo Grove, IL, USA).…”