Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Abdelhamid, Mahmoud A. S.; Rhind-Tutt, Alice V.; Ambrose, Benjamin; Craggs, Timothy D.

doi:10.3791/62378

Cited by 1 publication

(2 citation statements)

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“…Single-molecule confocal microscopy data were collected on the custom-built smfBox. Full details of the construction and operation of the instrument are described elsewhere including a step-by-step method protocol . Briefly, the smfBox alternates two lasers (515 nm, ∼220 μW and 635 nm, ∼70 μW, Omicron LuxX Plus lasers, powers measured immediately before the excitation dichroic) by TTL-controlled modulation of electronic shutters.…”

Section: Methodsmentioning

confidence: 99%

“…Full details of the construction and operation of the instrument are described elsewhere 31 including a step-by-step method protocol. 66 Briefly, the smfBox alternates two lasers (515 nm, ∼220 μW and 635 nm, ∼70 μW, Omicron LuxX Plus lasers, powers measured immediately before the excitation dichroic) by TTL-controlled modulation of electronic shutters. The beams are coupled into a single-mode fiber before being collimated (to 10 mm) and cropped by an iris (to 5 mm), then directed into a custom-built anodized-aluminum microscope body.…”

Section: Methodsmentioning

confidence: 99%

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High-Force Application by a Nanoscale DNA Force Spectrometer

Darcy¹,

Crocker²,

Wang³

et al. 2022

ACS Nano

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The ability to apply and measure high forces (>10 pN) on the nanometer scale is critical to the development of nanomedicine, molecular robotics, and the understanding of biological processes such as chromatin condensation, membrane deformation, and viral packaging. Established force spectroscopy techniques including optical traps, magnetic tweezers, and atomic force microscopy rely on micron-sized or larger handles to apply forces, limiting their applications within constrained geometries including cellular environments and nanofluidic devices. A promising alternative to these approaches is DNA-based molecular calipers. However, this approach is currently limited to forces on the scale of a few piconewtons. To study the force application capabilities of DNA devices, we implemented DNA origami nanocalipers with tunable mechanical properties in a geometry that allows application of force to rupture a DNA duplex. We integrated static and dynamic single-molecule characterization methods and statistical mechanical modeling to quantify the device properties including force output and dynamic range. We found that the thermally driven dynamics of the device are capable of applying forces of at least 20 piconewtons with a nanometer-scale dynamic range. These characteristics could eventually be used to study other biomolecular processes such as protein unfolding or to control high-affinity interactions in nanomechanical devices or molecular robots.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%