Tethered particle motion with single DNA molecules

Song, Dan; Mousley, Briana; Gambino, Stefano; Helou, Elsie; Loparo, Joseph J.; Price, Allen C.

doi:10.1119/1.4902187

Cited by 13 publications

(11 citation statements)

References 20 publications

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“…Tethered particle motion has been used by others to measure the persistence length of DNAs (33). Previously, we have used the technique to measure the persistence length of l DNAs end-labeled with a bead (diameter 1 mm) (39). A detailed description of the experimental methods can be found in the Supporting Material.…”

Section: Application Of Theory To Experimentsmentioning

confidence: 99%

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Price

Pilkiewicz

Graham

et al. 2015

Biophysical Journal

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Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length.

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Section: Application Of Theory To Experimentsmentioning

confidence: 99%

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Price

Pilkiewicz

Graham

et al. 2015

Biophysical Journal

Self Cite

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“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail ( 2 , 3 , 4 , 5 ). In addition, interactions with proteins such as DNA compaction by histones in eukaryotic chromatin ( 6 , 7 , 8 , 9 , 10 , 11 ) and prokaryotic architectural proteins ( 12 , 13 , 14 , 15 , 16 ), supercoiling ( 17 , 18 , 19 , 20 ), and repair processes ( 21 , 22 , 23 ) were extensively studied with magnetic tweezers (MT), optical tweezers (OT), acoustic force spectroscopy (AFS) ( 24 , 25 , 26 ), or tethered particle motion (TPM) ( 13 , 27 , 28 ). These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures such as extracellular protein collagen ( 29 , 30 , 31 ) or even entire cells ( 32 ).…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2020

Biophysical Journal

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead's position with nanometer accuracy in three dimensions but is computationally expensive. Realtime multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 mm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

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“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail [2][3][4][5]. In addition, the interactions with proteins, like the DNA compaction by histones in eukaryotic chromatin [6][7][8][9][10][11] and prokaryotic architectural proteins [12][13][14][15][16], supercoiling [17][18][19][20], and repair processes [21][22][23] were extensively studied with magnetic tweezers (MT) or optical tweezers (OT), Acoustic Force Spectroscopy (AFS) [24][25][26] or tethered particle motion (TPM) [13,27,28]. These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures, such as extracellular protein collagen [29][30][31], or even entire cells [32].…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2019

Preprint

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in arange of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required. SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10's of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

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Tethered particle motion with single DNA molecules

Cited by 13 publications

References 20 publications

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

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