High accuracy FIONA–AFM hybrid imaging

Multi-protein complexes are ubiquitous and play essential roles in many biological mechanisms. Single molecule imaging techniques such as electron microscopy (EM) and atomic force microscopy (AFM) are powerful methods for characterizing the structural properties of multi-protein and multi-protein–DNA complexes. However, a significant limitation to these techniques is the ability to distinguish different proteins from one another. Here, we combine high resolution fluorescence microscopy and AFM (FIONA–AFM) to allow the identification of different proteins in such complexes. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, we are able to align fluorescence and AFM information to ≥8 nm accuracy. This accuracy is sufficient to identify individual fluorescently labeled proteins in most multi-protein complexes. We investigate the limitations of localization precision and accuracy in fluorescence and AFM images separately and their effects on the overall registration accuracy of FIONA–AFM hybrid images. This combination of the two orthogonal techniques (FIONA and AFM) opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5–10 nm) information about the conformational properties of multi-protein complexes and the fluorescence can indicate spatial relationships of the proteins in the complexes.

Research highlights► Integration of fluorescent signals in AFM topography with high (<10 nm) accuracy. ► Investigation of limitations and quantitative analysis of fluorescence-AFM image registration using quantum dots. ► Fluorescence center tracking and display as localization probability distributions in AFM topography (FIONA–AFM). ► Application of FIONA–AFM to a biological sample containing damaged DNA and the DNA repair proteins UvrA and UvrB conjugated to quantum dots.