Determination of motility forces on isolated chromosomes with laser tweezers.
ABSTRACT: Quantitative determination of the motility forces of chromosomes during cell division is fundamental to understanding a process that is universal among eukaryotic organisms. Using an optical tweezers system, isolated mammalian chromosomes were held in a 1064 nm laser trap. The minimum force required to move a single chromosome was determined to be ? 0.8-5 pN. The maximum transverse trapping efficiency of the isolated chromosomes was calculated as ? 0.01-0.02. These results confirm theoretical force calculations of ? 0.1-12 pN to move a chromosome on the mitotic or meiotic spindle. The verification of these results was carried out by calibration of the optical tweezers when trapping microspheres with a diameter of 4.5-15 µm in media with 1-7 cP viscosity. The results of the chromosome and microsphere trapping experiments agree with optical models developed to simulate trapping of cylindrical and spherical specimens.
Project description:Acoustical tweezers open major prospects in microbiology for cells and microorganisms contactless manipulation, organization and mechanical properties testing since they are biocompatible, label-free and have the potential to exert forces several orders of magnitude larger than their optical counterpart at equivalent power. Yet, these perspectives have so far been hindered by the absence of spatial selectivity of existing acoustical tweezers - i.e., the ability to select and move objects individually - and/or their limited resolution restricting their use to large particle manipulation only and/or finally the limited forces that they could apply. Here, we report precise selective manipulation and positioning of individual human cells in a standard microscopy environment with trapping forces up to ~200 pN without altering their viability. These results are obtained with miniaturized acoustical tweezers combining holography with active materials to synthesize specific wavefields called focused acoustical vortices designed to produce stiff localized traps with reduced acoustic power.
Project description:Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.
Project description:Optical trapping forces depend on the difference between the trap wavelength and the extinction resonances of trapped particles. This leads to a wavelength-dependent trapping force, which should allow for the optimization of optical tweezers systems, simply by choosing the best trapping wavelength for a given application. Here we present an optical tweezer system with wavelength tunability, for the study of resonance effects. With this system, the optical trap stiffness is measured for single trapped particles that exhibit either single or multiple extinction resonances. We include discussions of wavelength-dependent effects, such as changes in temperature, and how to measure them.
Project description:Acoustic tweezers use sound radiation forces to manipulate matter without contact. They provide unique characteristics compared with the more established optical tweezers, such as higher trapping forces per unit input power and the ability to manipulate objects from the micrometer to the centimeter scale. They also enable the trapping of a wide range of sample materials in various media. A dramatic advancement in optical tweezers was the development of holographic optical tweezers (HOT) which enabled the independent manipulation of multiple particles leading to applications such as the assembly of 3D microstructures and the probing of soft matter. Now, 20 years after the development of HOT, we present the realization of holographic acoustic tweezers (HAT). We experimentally demonstrate a 40-kHz airborne HAT system implemented using two 256-emitter phased arrays and manipulate individually up to 25 millimetric particles simultaneously. We show that the maximum trapping forces are achieved once the emitting array satisfies Nyquist sampling and an emission phase discretization below ?/8 radians. When considered on the scale of a wavelength, HAT provides similar manipulation capabilities as HOT while retaining its unique characteristics. The examples shown here suggest the future use of HAT for novel forms of displays in which the objects are made of physical levitating voxels, assembly processes in the micrometer and millimetric scale, as well as positioning and orientation of multiple objects which could lead to biomedical applications.
Project description:Optical manipulation and label-free characterization of nanoscale structures open up new possibilities for assembly and control of nanodevices and biomolecules. Optical tweezers integrated with Raman spectroscopy allows analyzing a single trapped particle, but is generally less effective for individual nanoparticles. The main challenge is the weak gradient force on nanoparticles that is insufficient to overcome the destabilizing effect of scattering force and Brownian motion. Here, we present standing-wave Raman tweezers for stable trapping and sensitive characterization of single isolated nanostructures with a low laser power by combining a standing-wave optical trap with confocal Raman spectroscopy. This scheme has stronger intensity gradients and balanced scattering forces, and thus can be used to analyze many nanoparticles that cannot be measured with single-beam Raman tweezers, including individual single-walled carbon nanotubes (SWCNT), graphene flakes, biological particles, SERS-active metal nanoparticles, and high-refractive semiconductor nanoparticles. This would enable sorting and characterization of specific SWCNTs and other nanoparticles based on their increased Raman fingerprints.
Project description:Plasmonic optical tweezers are a ubiquitous tool for the precise manipulation of nanoparticles and biomolecules at low photon flux, while femtosecond-laser optical tweezers can probe the nonlinear optical properties of the trapped species with applications in biological diagnostics. In order to adopt plasmonic optical tweezers in real-world applications, it is essential to develop large-scale fabrication processes without compromising the trapping efficiency. Here, we develop a novel platform for continuous wave (CW) and femtosecond plasmonic optical tweezers, based on gold-coated black silicon. In contrast with traditional lithographic methods, the fabrication method relies on simple, single-step, maskless tabletop laser processing of silicon in water that facilitates scalability. Gold-coated black silicon supports repeatable trapping efficiencies comparable to the highest ones reported to date. From a more fundamental aspect, a plasmon-mediated efficiency enhancement is a resonant effect, and therefore, dependent on the wavelength of the trapping beam. Surprisingly, a wavelength characterization of plasmon-enhanced trapping efficiencies has evaded the literature. Here, we exploit the repeatability of the recorded trapping efficiency, offered by the gold-coated black silicon platform, and perform a wavelength-dependent characterization of the trapping process, revealing the resonant character of the trapping efficiency maxima. Gold-coated black silicon is a promising platform for large-scale parallel trapping applications that will broaden the range of optical manipulation in nanoengineering, biology, and the study of collective biophotonic effects.
Project description:The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the ?-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.
Project description:The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.
Project description:Manipulation of colloidal objects with light is important in diverse fields. While performance of traditional optical tweezers is restricted by the diffraction-limit, recent approaches based on plasmonic tweezers allow higher trapping efficiency at lower optical powers but suffer from the disadvantage that plasmonic nanostructures are fixed in space, which limits the speed and versatility of the trapping process. As we show here, plasmonic nanodisks fabricated over dielectric microrods provide a promising approach toward optical nanomanipulation: these hybrid structures can be maneuvered by conventional optical tweezers and simultaneously generate strongly confined optical near-fields in their vicinity, functioning as near-field traps themselves for colloids as small as 40 nm. The colloidal tweezers can be used to transport nanoscale cargo even in ionic solutions at optical intensities lower than the damage threshold of living micro-organisms, and in addition, allow parallel and independently controlled manipulation of different types of colloids, including fluorescent nanodiamonds and magnetic nanoparticles.
Project description:Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.