Double Optical Tweezers Research Articles

Classification of single extracellular vesicles in a double nanohole optical tweezer for cancer detection

A major challenge in cancer prognostics is finding early biomarkers that can accurately identify cancer. Circulating tumor cells are rare and circulating tumor DNA can not provide information about the originating cell. Extracellular vesicles (EVs) contain cell specific information, are abundant in fluids, and have unique properties between cancerous and non-cancerous. Fluorescence measurements have limitations from intrinsic fluorescent background signals, photobleaching, non-specific labelling, and EV structural modifications. Here, we demonstrate a label-free approach to classification of 3 different EVs, derived from non-malignant, non-invasive cancerous, and invasive cancerous cell lines. Using double nanohole optical tweezers, the scattering from single trapped EVs is measured, and using a 1D convolutional neural network, we are able to classify the time series optical signal into its respective EV class with greater than 90% accuracy.

Optical trapping of photochromic microcrystals by a dual fiber tweezers

Upon light irradiation, photochromic materials exhibit drastic changes in absorbance in addition to molecular and mechanical changes. In this study, we demonstrated optical trapping of photochromic microcrystals in a double fiber optical tweezers system combined with two external lights for photoisomerization and observed changes in the trapping force by photoisomerization. In situ photoisomerizations of the single crystal trapped in the system were performed thrice revealing a reversible change in the trapping force. The trapping stiffness for the colored crystal was one-third of that for the decolorized crystal in the axial direction, whereas almost no difference was observed in the transverse direction. Furthermore, based on the intermittent motion and asymmetry of the positional distribution of the trapped crystals, we inferred that the motion includes transitions through multiple stable sites, and the ease of the transition is related to the isomerization state of the crystals. Such switching of optical trapping indicates the possibility of revealing the physical properties of trapped materials through trapping schemes, and the nonlinearity in the trapping is also expected to be applied to advanced computing with physical phenomena.

Isolating and enhancing single-photon emitters for 1550nm quantum light sources using double nanohole optical tweezers.

Single-photon sources are required for quantum technologies and can be created from individual atoms and atom-like defects. Erbium ions produce single photons at low-loss fiber optic wavelengths, but they have low emission rates, making them challenging to isolate reliably. Here, we tune the size of gold double nanoholes (DNHs) to enhance the emission of single erbium emitters, achieving 50× enhancement over rectangular apertures previously demonstrated. This produces enough enhancement to show emission from single nanocrystals at wavelengths not seen in our previous work, i.e., 400 and 1550nm. We observe discrete levels of emission for nanocrystals with low numbers of emitters and demonstrate isolating single emitters. We describe how the trapping time is proportional to the enhancement factor for a given DNH structure, giving us an independent way to measure the enhancement. This shows a promising path to achieving single emitter sources at 1550nm.

Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly.

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.

Analysis of Egg White Protein Composition with Double Nanohole Optical Tweezers.

We use a double nanohole optical tweezer to analyze the protein composition of egg white through analysis of many individual protein trapping events. The proteins are grouped by mass based on two metrics: standard deviation of the trapping laser intensity fluctuations from the protein diffusion and the time constant of these fluctuations coming from the autocorrelation. Quantitative analysis is demonstrated for artificial samples, and then, the approach is applied to real egg white. The composition found from real egg white corresponds well to past reports using gel electrophoresis. This approach differs from past works by allowing for individual protein analysis in heterogeneous solutions without the need for denaturing, labeling, or tethering.

Toward Controlled Photothermal Treatment of Single Cell: Optically Induced Heating and Remote Temperature Monitoring In Vitro through Double Wavelength Optical Tweezers

Cancer treatment based on hyperthermia (HT) relies on exposing the malignant cells to elevated local temperature. Although the procedure has been successfully applied in clinics, the fundamental aspects of HT are not yet fully understood. In order to verify the susceptibility of single cells in vitro to raised temperature, we have developed novel nano- and microtools. In particular, an optical double-trap system utilizing combined galvano-mirror scanning and spatial light phase modulator was devised to manipulate several micron-sized objects simultaneously. The manipulation comprised both optical trapping and translocating, on demand photoactivated heating, and simultaneous remote temperature readout of living cells, infrared activated heaters and optical thermometers, respectively. Mesoporous silicon microparticles were used as an infrared absorber to generate an increased temperature of about 100 °C with 0.4 W laser power. The optical micron-sized thermometer was based on up-converting Yb–Er codoped nan...

Low cost double optical tweezers to manipulate microorganisms and micro-crystals

An inexpensive double optical tweezers has been used for trapping and manipulating microorganisms and micro-sized crystals with regular and irregular shapes. The intensity and spatial location of the traps were controlled through mirror displacements devices. A stable infrared laser beam with tunable power was used. The particles were manipulated and observed in an inverted microscope adjusted to locate the infrared trapping laser beam into the holder. A bacilliform bacterium with an approximate size of 12.0µm was captured by its two elongated ends, exerting control of their displacements and rotations. Also, regular micro-crystals measuring approximately 10.0µmx4.0µmx0.5µm were captured, achieving nanometric control of their displacements and rotations in two dimensions, parallel to one of their faces

Real-time force measurement in double wavelength optical tweezers

In optical tweezers, the trap stiffness varies across the sample area. To avoid this problem, the force measurement is often performed in a fixed place where the trap stiffness is well determined. However, for some experiments, bringing the sample to the fixed position is problematic. In this paper, we describe a precise and fast procedure for mapping the trap stiffness over the whole sample area. Such a map allows development of a real-time procedure for force measurement at any point of the sample area. The presented method is particularly suitable for measuring forces, for example, in living cells samples.

Real-time force measurement in double wavelength optical tweezers

Real-time force measurement in double wavelength optical tweezers

Characterization of Individual Magnetic Nanoparticles in Solution by Double Nanohole Optical Tweezers.

We study individual superparamagnetic Fe3O4 (magnetite) nanoparticles in solution using a double nanohole optical tweezer with magnetic force setup. By analysis of the trapping optical transmission signal (step size, autocorrelation, the root-mean-square signal, and the distribution with applied magnetic field), we are able to measure the refractive index, magnetic susceptibility, remanence and size of each trapped nanoparticle. The size distribution is found to agree well with scanning electron microscopy measurements, and the permeability, magnetic susceptibility and remanence values are all in agreement with published results. Our approach demonstrates the versatility of the optical tweezer with magnetic field setup to characterize nanoparticles in fluidic mixtures with potential for isolation of desired particles and pick-and-place functionality.

Characterization at the individual cell level and in whole blood samples of shear stress preventing red blood cells aggregation

The aggregation of red blood cells (RBC) is an intrinsic feature of blood that has a strong impact on its microcirculation. For a number of years it has been attracting a great attention in basic research and clinical studies. Here, we study a relationship between the RBC aggregation parameters measured at the individual cell level and in a whole blood sample. The home made optical tweezers were used to measure the aggregating and disaggregating forces for a pair of interacting RBCs, at the individual cell level, in order to evaluate the corresponding shear stresses. The RheoScan aggregometer was used for the measurements of critical shear stress (CSS) in whole blood samples. The correlation between CSS and the shear stress required to stop an RBC pair from aggregating was found. The shear stress required to disaggregate a pair of RBCs using the double channel optical tweezers appeared to be about 10 times higher than CSS. The correlation between shear stresses required to prevent RBCs from aggregation at the individual cell level and in whole blood samples was estimated and assessed quantitatively. The experimental approach developed has a high potential for advancing hemorheological studies.

Photonic nanojets in optical tweezers

Photonic nanojets have been brought into attention ten years ago for potential application in ultramicroscopy, because of its sub-wavelength resolution that can enhance detection and interaction with matter. For these novel applications under development, the optical trapping of a sphere acts as an ideal framework to employ photonic nanojets. In the present study, we generated nanojets by using a highly focused incident beam, in contrast to traditional plane waves. The method inherits the advantage of optical trapping, especially for intracellular applications, with the microsphere in equilibrium on the beam propagation axis and positioned arbitrarily in space. Moreover, owing to optical scattering forces, when the sphere is in equilibrium, its center shifts with respect to the focal point of the incident beam. However, when the system is in stable equilibrium with a configuration involving optical tweezers, photonic nanojets cannot be formed. To overcome this issue, we employed double optical tweezers in an unorthodox configuration involving two collinear and co-propagating beams, the precise positioning of which would turn on/off the photonic nanojets, thereby improving the applicability of photonic nanojets.

Measuring red blood cell aggregation forces using double optical tweezers

Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10−3 (0.1–2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10−3 (1.0–4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.

Single Molecule Tracking of Lac Repressor Diffusing on Stretched DNA

Lac Repressor (LacI) is a DNA-binding protein that regulates genes expression by specific binding to its target sequence on DNA. This sequence is found rapidly among millions of base pairs in the genome, presumably via a combination of 3D and 1D-diffusion (following the interaction with non-specific DNA). Here we report diffusion analysis through the combination of single molecule localization and manipulation. For single molecule localization, Atto532 dye has been covalently linked to a single-cysteine mutant of LacI, LacIQ231C. A double optical tweezers system is used for trapping and stretching a single DNA molecule. All components of the experiment (protein, DNA, beads and buffer) are assembled (and/or rapidly exchanged) with a flow-system. This integrated configuration has several advantages: biomolecules are not in the proximity of the glass surface thus preventing possible electrostatic effects; controlled forces can be measured or applied. We characterized LacI 1D-diffusion under different forces applied to the DNA. The figure shows an example of the data obtained in the form of a kymogram.Acknowledgments:The research leading to the results has received funding from Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAXView Large Image | View Hi-Res Image | Download PowerPoint Slide

Cellular viscoelasticity probed by active rheology in optical tweezers

A novel approach to probe viscoelastic properties of cells based on double trap optical tweezers is reported. Frequency dependence of the tangent of phase difference in the movement of the opposite erythrocyte edges while one of the edges is forced to oscillate by optical tweezers appeared to be highly dependent on the rigidity of the cellular membrane. Effective viscoelastic parameters characterizing red blood cells with different stiffnesses (normal and glutaraldehyde-fixed) are determined. It is shown that the photo-induced effects caused by laser trapping at the power level used in the experiments are negligible giving the possibility to use the offered technique for dynamic monitoring of soft materials viscoelastic properties.

Normal and system lupus erythematosus red blood cell interactions studied by double trap optical tweezers: direct measurements of aggregation forces

Direct measurements of aggregation forces in piconewton range between two red blood cells in pair rouleau are performed under physiological conditions using double trap optical tweezers. Aggregation and disaggregation properties of healthy and pathologic (system lupus erythematosis) blood samples are analyzed. Strong difference in aggregation speed and behavior is revealed using the offered method which is proposed to be a promising tool for SLE monitoring at single cell level.

Investigating the Interaction of Single UvrA Dimers with DNA Using a Combination of Fluorescent Microscopy and Optical Tweezers

Nucleotide excision repair (NER) is a DNA repair mechanism responsible for replacement of base pairs damaged e.g. by exposure to UV light. In E. coli, NER is initiated by the UvrA-dimer, which searches for and localizes damaged nucleotides to which then the UvrB-dimer is recruited. We study the damage search of UvrA by visualizing single, fluorescently labeled UvrA-dimers interacting with a DNA molecule manipulated by double optical tweezers. This approach enables us to observe UvrA binding in real time to DNA under different tensions.We find that, in the presence of ATP, the UvrA-dimer displays static, non-specific DNA binding of long duration (>10s), in accord with a recent publication [1]. In addition, our results reveal that non-specific UvrA binding is tension dependent: Binding events are prolonged on DNA under tension, indicating that the dissociation rate decreases while the association rate remains rather constant, thus supporting a model in which UvrA binding unwinds DNA [2].Finally, we observe that the duration of binding events decreases by several orders of magnitude if ADP instead of ATP is present in the buffer (<<1s). Binding is sensitive to tension, even more so than in the ATP experiment, resulting in a more than tenfold increase of the duration of binding events when DNA tension is increased from 5 to 65pN. In addition, our results demonstrate that UvrA has a higher binding affinity for ADP than for ATP. We are currently investigating whether ADP binding plays an important role in accelerating the DNA damage search in vivo.[1] Kad, N. M., Wang, H., Kennedey, G. G., Warshaw, D. M., van Houten, B. (2010). Mol. Cell 37, 702–713.[2] Oh, E., Grossman, L. (1986). Nucleic Acids Res. 14, 8557–8571.

Archaeal DNA Organization: The Mechanism of Alba I & II Revealed

Throughout the kingdoms of life cells face a similar problem, namely the size of their genome is very large compared to the volume of the cell. Although each organism employs its own set of proteins to compact their genome, up to a factor of 10.000, the method of compaction seems highly conserved. Besides, DNA organization is known to be a key regulatory mechanism involved in many important processes such as gene regulation and DNA replication. The protein Alba, one of the most abundant proteins in Archaea, has been suggested to play an important role in DNA organization. Previous studies have shown that Alba binds as a dimer to non-specific DNA sequences and is able to condense DNA. However, little is known about its mechanism and structural role in DNA-organization. Here we show, using several single-molecule imaging and manipulation techniques such as AFM, double and quadruple optical tweezers, the condensing and possible regulation mechanism of Alba. From the surprising structural changes to the protein-bound DNA the binding constant, footprint and cooperativity factor are obtained by using a McGhee von Hippel analysis. We furthermore prove that the Archaea protein Alba II, which is thought to have a regulatory function, controls the binding efficiency of Alba I by changing the cooperativity of the Alba & Alba II protein mix.

DNA Polymerization in Optical Tweezers

Molecular motors associated with nucleic acids ensure replication, maintenance and expression of genetic information, which is crucial for life. Biochemical and biological studies have generated considerable knowledge about these molecular motors. However, these studies assess only sum reactions/effects due to ensemble averaging. Single molecule (SM) measurements overcome this shortcoming. Such measurements allow learning about individual motor functions, such as processivity, fidelity, step size, template dependence and generated forces. Transient intermediate states and rarely occurring events can also be observed.We are developing an assay based on bacteriophage phi29 polymerase to study DNA/RNA polymerases in action using high resolution optical tweezers (OT) combined with fluorescence imaging (OT-SMF). We use an optically levitated ‘dumbbell’ assay: the nucleic acid (NA) construct features biotinylated and digixigeninated ends that tether two different kinds of microspheres (coated with streptavidin and anti-digoxigenin); the protein of interest is attached directly to the NA tether. Since in our double OT instrument one trap is stable whereas the other mobile, we can manipulate the tethers, detect changes in tether length and stiffness, apply different forces and simultaneously observe the fluorescently stained template/protein. We present preliminary results on DNA replication by phi29 polymerase.

Tracking of Single Quantum Dot Labeled EcoRV Sliding along DNA Manipulated by Double Optical Tweezers

Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.