Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy

Abstract

Correlative imaging by fluorescence and force microscopy is an emerging technology to acquire orthogonal information at the nanoscale. Whereas atomic force microscopy excels at resolving the envelope structure of nanoscale specimens, fluorescence microscopy can detect specific molecular labels, which enables the unambiguous recognition of molecules in a complex assembly. Whereas correlative imaging at the micrometer scale has been established, it remains challenging to push the technology to the single-molecule level. Here, we used an integrated setup to systematically evaluate the factors that influence the quality of correlative fluorescence and force microscopy. Optimized data processing to ensure accurate drift correction and high localization precision results in image registration accuracies of ∼25 nm on organic fluorophores, which represents a 2-fold improvement over the state of the art in correlative fluorescence and force microscopy. Furthermore, we could extend the Atto532 fluorophore bleaching time ∼2-fold, by chemical modification of the supporting mica surface. In turn, this enables probing the composition of macromolecular complexes by stepwise photobleaching with high confidence. We demonstrate the performance of our method by resolving the stoichiometry of molecular subpopulations in a heterogeneous EcoRV-DNA nucleoprotein ensemble.

How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

“…When imaging single molecules the challenge is amplified by the requirement of nanometer precision for localization. The use of fluorescent fiducials for image registration has been shown invaluable, 112,157,158 and after the development and validation of software routines for consistent, accurate and convenient image registration, AFM/TIRFM systems can be used in a routine manner. 155,156,[159][160][161] These tools have helped researchers to understand mechanistic aspect of the cellular DNA repair systems, like Homologous Recombination (HR), that efficiently restore genome integrity in healthy cells.…”

DNA Origami Fiducial for Accurate 3D Atomic Force Microscopy Imaging

“…We note that the design of the DNA origami structure could be altered or extended for specific purposes, for example, by addition of additional steps or attachment of fluorescent dyes. A combination with fluorescent markers has the potential to enable simultaneous use of the fiducial for tip shape and fluorescence calibration. − ,, Another research direction would be to go beyond imaging and to use the fiducial as a mechanical stiffness marker to study the compliance of biomolecules to indentation forces, e.g., to probe the effects of silicification , or other functionalizations. Soft biological materials are deformed by interactions with the AFM tip, and our fiducial structure could provide a convenient reference to take these effects into account while correcting images.…”

A DNA origami fiducial for accurate 3D AFM imaging

“…We note that the design of the DNA origami structure could be altered or extended for specific purposes, for example by addition of additional steps or attachment of fluorescent dyes. A combination with fluorescent markers has the potential to enable simultaneous use of the fiducial as a tip shape and fluorescence calibration 39-42, 58, 59 . Another research direction would be to go beyond imaging and to use the fiducial as a mechanical stiffness marker to study the compliance of biomolecules to indentation forces 60 , e.g.…”