High accuracy FIONA–AFM hybrid imaging

Fronczek, David Norman; Quammen, Cory; Wang, H.; Kisker, Caroline; Superfine, R.; Taylor, Russell M.; Erie, Dorothy A.; Teßmer, Ingrid

doi:10.1016/j.ultramic.2011.01.020

Cited by 27 publications

(35 citation statements)

References 20 publications

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“…This method is limited because identification of specific proteins in heterogeneous assemblies depends on distinct structural features, whereas most proteins have a similar globular shape. Molecular recognition in high-resolution SFM images can be achieved by combining SFM with a fluorescence microscope capable of single-fluorescence detection (4)(5)(6). By labeling individual proteins or single DNA molecules, it is possible to distinguish the position of different components in a highly resolved complex.…”

mentioning

confidence: 99%

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Sánchez¹,

Kertokalio²,

Rossum-Fikkert³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.DNA break repair | genome stability | DNA-protein interaction | image registration | single-molecule microscopy T o understand how proteins cooperate to perform elaborate genome transactions, we need to know how they are arranged relative to each other in functional complexes. Determining the structure of such complex and often variable assemblies is a challenge. The scanning force microscope (SFM) is ideal for visualizing DNA-protein assemblies under native conditions at a resolution limited by the radius of curvature of the scanning tip (usually 5-10 nm) (1-3). This method is limited because identification of specific proteins in heterogeneous assemblies depends on distinct structural features, whereas most proteins have a similar globular shape. Molecular recognition in high-resolution SFM images can be achieved by combining SFM with a fluorescence microscope capable of single-fluorescence detection (4-6). By labeling individual proteins or single DNA molecules, it is possible to distinguish the position of different components in a highly resolved complex.The multiprotein complexes of homologous recombination (HR) are an ideal example to establish methods for hybrid microscopy that can be validated on the basis of known structures and used to describe unknown arrangements. HR is a mesoscale DNA rearrangement process achieved by the coordinated action of several proteins and used for DNA repair (7,8). In the core reaction of HR, human recombinase RAD51-DNA filaments search and invade homologous undamaged dsDNA. After strand exchange between the invading ssDNA and its complement, the homologous strand of the duplex is used as template for repair synthesis. Genetic and biochemical evidence show that several mediator proteins, such as RAD54, are required to regulate RAD51 nucleoprotein filament function and pr...

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mentioning

confidence: 99%

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Sánchez¹,

Kertokalio²,

Rossum-Fikkert³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…This speed is approximately nine times faster than CPU correlation (averagely 140.5630.3 s per correlation), and about eleven times higher than manual operation (averagely 174.9656.4 s per correlation). The average drift rate of our nanomanipulation set-up was 1.24 nm s 21 , determined with the method we previously reported. 29 With GPUaccelerated ASIFT, a 15.3 s computation time translates to SEM image drift of ,19 nm.…”

Section: Resultsmentioning

confidence: 99%

“…These landmarks can be easily identified in images captured under different modes of microscopy (e.g., SEM and fluorescence). [18][19][20][21] However, our DNA extraction task must be conducted under specific SEM imaging conditions with limited imaging resolution, imposed by the necessity of preserving the biochemical integrity of the sample. Such poor imaging conditions make the identification of nanometer-sized fiducial marks difficult.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence and SEM correlative microscopy for nanomanipulation of subcellular structures

et al. 2014

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Nanomanipulation under scanning electron microscopy (SEM) enables direct interactions of a tool with a sample. We recently developed a nanomanipulation technique for the extraction and identification of DNA contained within sub-nuclear locations of a single cell nucleus. In nanomanipulation of sub-cellular structures, a key step is to identify targets of interest through correlating fluorescence and SEM images. The DNA extraction task must be conducted with low accelerating voltages resulting in low imaging resolutions. This is imposed by the necessity of preserving the biochemical integrity of the sample. Such poor imaging conditions make the identification of nanometer-sized fiducial marks difficult. This paper presents an affine scale-invariant feature transform (ASIFT) based method for correlating SEM images and fluorescence microscopy images. The performance of the image correlation approach under different noise levels and imaging magnifications was quantitatively evaluated. The optimal mean absolute error (MAE) of correlation results is 68634 nm under standard conditions. Compared with manual correlation by skilled operators, the automated correlation approach demonstrates a speed that is higher by an order of magnitude. With the SEM-fluorescence image correlation approach, targeted DNA was successfully extracted via nanomanipulation under SEM conditions.

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“…Hybrid methods combine AFM with fluorescence microscopy (Fronczek et al, 2011; Sanchez, Kanaar, & Wyman, 2010; Sanchez, Kertokalio, van Rossum-Fikkert, Kanaar, & Wyman, 2013). These techniques overcome a serious limitation of AFM (and electron microscopy) to distinguish different proteins.…”

Section: Complementary Techniquesmentioning

confidence: 99%

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

LeBlanc

Wilkins

et al. 2017

Methods in Enzymology

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Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein–protein and protein–DNA interactions. It can be used to capture snapshots of protein–DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein–protein and protein–DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein–protein and protein–DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein–DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein–protein and protein–nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure–function relationships that govern DNA repair processes.

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High accuracy FIONA–AFM hybrid imaging

Cited by 27 publications

References 20 publications

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Fluorescence and SEM correlative microscopy for nanomanipulation of subcellular structures

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

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