121 research outputs found
Position clamping in a holographic counterpropagating optical trap
Optical traps consisting of two counterpropagating, divergent beams of light allow relatively high forces to be exerted along the optical axis by turning off one beam, however the axial stiffness of the trap is generally low due to the lower numerical apertures typically used. Using a high speed spatial light modulator and CMOS camera, we demonstrate 3D servocontrol of a trapped particle, increasing the stiffness from 0.004 to 1.5ÎŒNm<sup>â1</sup>. This is achieved in the âmacro-tweezersâ geometry [Thalhammer, J. Opt. 13, 044024 (2011); Pitzek, Opt. Express 17, 19414 (2009)], which has a much larger field of view and working distance than single-beam tweezers due to its lower numerical aperture requirements. Using a 10Ă, 0.2NA objective, active feedback produces a trap with similar effective stiffness to a conventional single-beam gradient trap, of order 1ÎŒNm<sup>â1</sup> in 3D. Our control loop has a round-trip latency of 10ms, leading to a resonance at 20Hz. This is sufficient bandwidth to reduce the position fluctuations of a 10ÎŒm bead due to Brownian motion by two orders of magnitude. This approach can be trivially extended to multiple particles, and we show three simultaneously position-clamped beads
Nano handling and measurement of biological cells in culture
A thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of PhilosophyThis thesis systematically investigates the nano handling and measurement techniques for biological cells in culture and studies the techniques to realize innovative and multi-functional applications in biomedicine. Among them, the technique based on AFM is able to visualize and quantify the dynamics of organic cells in culture on the nano scale. Especially, the cellular shear adhesion force on the various locations of biological cells was firstly accurately measured in the research of the cell-substrate interaction in terms of biophysical perspective. The innovative findings are conductive to study the cell-cell adhesion, the cell-matrix adhesion which is related to the cell morphology structure, function, deformation ability and adhesion of cells and better understand the cellular dynamic behaviors. Herein, a new liquid-AFM probe unit and an increment PID control algorithm were implemented suitable for scanning the cell samples under the air conditions and the liquid environments. The influence between the surface of sample and
the probe, and the damage of probe during the sample scanning were reduced. The proposed system is useful for the nano handling and measurement of living cells.
Besides, Besides, to overcome the limitations of liquid-AFMs, the multiple optical tweezers were developed to integrate with the liquid-AFM. The technique based on laser interference is able to characterize the optical trap stiffness and the escape velocity, especially to realize the capture and sorting of multiple cells by a polarization-controlled periodic laser interference. It can trap and move hundreds of cells without physical contact, and has broad application prospects in cytology. Herein, a new experimental method integrated with the positioning analysis in the Z direction was used to improve the fluid force method for the calibration and characterize the mechanical forces exerted on optical traps and living cells. Moreover, a sensitive and highly efficient polarization-controlled three-beam interference set-up was developed for the capture and sorting of multiple cells. By controlling the polarization angles of the beams, various intensity distributions and different sizes of dots were obtained. Subsequently, we have experimentally observed multiple optical tweezers and the sorting of cells with different polarization angles, which are in accordance with the theoretical analysis
Stretching single DNA-molecules with temperature-stabilized optical tweezers
This thesis consists of two parts; in the first part we performed a single-molecule force extension measurement with 10kb long DNA-molecules from phage-λ to validate the calibration and single-molecule capability of our optical tweezers instrument. Fitting the worm-like chain interpolation formula to the data revealed that ca. 71% of the DNA tethers featured a contour length within ±15% of the expected value (3.38 ”m). Only 25% of the found DNA had a persistence length between 30 and 60 nm. The correct value should be within 40 to 60 nm. In the second part we designed and built a precise temperature controller to remove thermal fluctuations that cause drifting of the optical trap. The controller uses feed-forward and PID (proportional-integral-derivative) feedback to achieve 1.58 mK precision and 0.3 K absolute accuracy. During a 5 min test run it reduced drifting of the trap from 1.4 nm/min in open-loop to 0.6 nm/min in closed-loop.TÀmÀ tutkielma koostuu kahdesta osasta; ensimmÀisessÀ osassa tutkimme optinen pinsetti-laitteistomme kalibraatiota ja soveltuvuutta yksittÀismolekyylikokeisiin 10kb pituisien λ-faagista perÀisin olevien DNA-molekyylien voima-venytyskokeilla. Mittauksiin sovitettiin 'worm-like chain'-interpolaatio malli, joka osoitti, ettÀ n. 71%:lla löydetyistÀ DNA ketjuista oli pituus 15% sisÀllÀ odotetusta pituudesta (3.38 ”m). Vain 25%:lla DNA:sta oli sitkeyspituus 30-60 nm odotetun arvon ollessa 40-60 nm. Tutkielman toisessa osassa rakensimme lÀmpötilakontrollerin, jonka tarkoituksena oli poistaa lÀmpötilavaihteluiden aiheuttama 'ajelehtiminen' optisilla pinseteillÀ kiinnipidetyn mikroskooppisen pallon paikassa. Kontrolleri kÀyttÀÀ 'feedforward-' ja takaisinkytkentÀsilmukoita saavuttaakseen 1.58 mK sisÀisen tarkkuuden ja 0.3 K ulkoisen tarkkuuden. Viiden minuutin kokeen aikana pallo ajelehti 1.4 nm/min avoimella silmukalla ja 0.6 nm/min suljetulla silmukalla
Modeling and Experimental Techniques to Demonstrate Nanomanipulation With Optical Tweezers
The development of truly three-dimensional nanodevices is currently impeded by the absence of effective prototyping tools at the nanoscale. Optical trapping is well established for flexible three-dimensional manipulation of components at the microscale. However, it has so far not been demonstrated to confine nanoparticles, for long enough time to be useful in nanoassembly applications. Therefore, as part of this work we demonstrate new techniques that successfully extend optical trapping to nanoscale manipulation.
In order to extend optical trapping to the nanoscale, we must overcome certain challenges. For the same incident beam power, the optical binding forces acting on a nanoparticle within an optical trap are very weak, in comparison with forces acting on microscale particles. Consequently, due to Brownian motion, the nanoparticle often exits the trap in a very short period of time. We improve the performance of optical traps at the nanoscale by using closed-loop control. Furthermore, we show through laboratory experiments that we are able to localize nanoparticles to the trap using control systems, for sufficient time to be useful in nanoassembly applications, conditions under which a static trap set to the same power as the controller is unable to confine a same-sized particle.
Before controlled optical trapping can be demonstrated in the laboratory, key tools must first be developed. We implement Langevin dynamics simulations to model the interaction of nanoparticles with an optical trap. Physically accurate simulations provide a robust platform to test new methods to characterize and improve the performance of optical tweezers at the nanoscale, but depend on accurate trapping force models. Therefore, we have also developed two new laboratory-based force measurement techniques that overcome the drawbacks of conventional force measurements, which do not accurately account for the weak interaction of nanoparticles in an optical trap. Finally, we use numerical simulations to develop new control algorithms that demonstrate significantly enhanced trapping of nanoparticles and implement these techniques in the laboratory.
The algorithms and characterization tools developed as part of this work will allow the development of optical trapping instruments that can confine nanoparticles for longer periods of time than is currently possible, for a given beam power. Furthermore, the low average power achieved by the controller makes this technique especially suitable to manipulate biological specimens, but is also generally beneficial to nanoscale prototyping applications. Therefore, capabilities developed as part of this work, and the technology that results from it may enable the prototyping of three-dimensional nanodevices, critically required in many applications
Optical tweezers combined with interference reflection microscopy for quantitative trapping and 3D imaging
Optical tweezers are an indispensable tool in biophysical single-molecule studies. They
provide the ability to mechanically probe the characteristics of biological processes, such
as active transport of cargo by molecular motors. To this end, functionalized (sub)micron-
sized dielectric particles are held in a tightly focused laser trap while external forces lead
to displacements of the particle from the trap center. The measurement and calibration of
these displacements yield insights into the mechanical properties of the molecule of interest.
The study of molecular motors, such as kinesins, is carried out in in vitro surface-based
experimental assays. The experimental needs for such assays are challenging. The instrument
must be stabilized, i.e. decoupled form external noise, and drift must be minimized, and it
needs to be combined with state of the art microscopy techniques to visualize the sample.
These are, on the one hand, single-molecule fluorescence detection and, on the other hand,
robust label-free imaging of diffraction limited specimen. The latter is commonly realized
by differential interference contrast (DIC) microscopy, which is an expensive and rather
complicated technique that also restricts the design of the optical tweezers and, therefore,
reduces the experimental possibilities.
Optical tweezers experiments, moreover, rely on precise and reliable calibration. Despite
its importance, calibration is, at times, carried out with obsolete methods or based on vague
assumptions. Especially, in the vicinity of the sample surface, where hydrodynamic effects
can have a significant influence, such assumptions fail largely. Here, height-dependent active
power spectral density analysis of the Brownian motion of the trapped particle can ame-
liorate these inaccuracies, butâcompared to other methodsâis rather cumbersome, time-
consuming and easy-to-use solutions are lacking.
In this work I designed and assembled an optical tweezers setup combined with total in-
ternal reflection fluorescence (TIRF) microscopy. Furthermore, I succeeded to reduce design
restrictions of the optical tweezers by combining it with interference reflection microscopy,
which is a simple, cost-efficient and robust contrast technique that can visualize diffraction
limited specimen in three dimensions, such as microtubules. Moreover, I was able to use this
technique to determine the three-dimensional profile of an upward bent microtubule which I
used to simultaneously calibrate the evanescent field depth of the TIRF microscope. In ad-
dition, I programmed a free and open-source optical tweezers calibration software, PyOTC,
that provides the means for height-dependent active power spectral density analysis.
My work will possibly influence the design of optical tweezers instruments for surface-based
experiments. LED-based IRM could further improve or complement label-free detection tech-
niques such as interferometric scattering microscopy. The free and open-source calibration
software package could help to precisely calibrate optical tweezers data. Moreover, because
the source is available to anybody, calibration and therefore the analysis of optical tweezers
data will be more transparent to the scientific community
Miniaturised magnetic bead actuator-based atomic force microscope for single-molecule measurements
Single molecular techniques have been providing researchers powerful tools to reveal the mechanisms of bioprocesses by investigating the behaviours and properties of individual molecules. Itâs also an essential way to study the functional differences and accesses the parameters of individual molecules.
Atomic force microscopy (AFM) is one of the most popular technologies to probe into individual molecules and has provided insights into structure, kinetics and dynamics of many molecules. However, the conventional AFM use cantilever-based sensors and piezo-based actuators which are relatively large in dimension and prone to drift and noise.
This thesis focuses on the development of a customised AFM for single molecule force spectroscopy experiments which is capable of both magnetic and piezo actuation. The magnetic actuation method unitises miniaturise magnetic beads as actuators reduces the actuator size significantly and performs experiment in non-contact way, thus reduces the impact of noise and drift. The resolution of the setup is verified experimentally and comparable to commercial AFM in single molecule force spectroscopy applications.
Single molecule force spectroscopy experiments using both varying loading rates and force clamp methods have been performed using biotin-streptavidin and heparin-FGF2 molecule pairs. The energy landscapes of their bonds have been studied
Optical and magnetic tweezers for applications in single-molecule biophysics and nanotechnology
Tesis doctoral inĂ©dita leĂda en la Universidad AutĂłnoma de Madrid, Facultad de Ciencias, Departamento de FĂsica de la Materia Condensada. Fecha de lectura: 22-01-201
Single biomolecule studies using optical tweezers
Single biological molecule studies enable to probe and visualize exciting details of the
events in physiological in vivo processes. The basic underlying question of this
dissertation is to understand biological processes at a single molecule level. In
contrast to ensemble techniques, advances in single molecule manipulation (e.g.
optical and magnetic tweezers, atomic force microscopy) and / or fluorescence
techniques allow to investigate the properties of individual molecules in real time with
a possibility to change external conditions (buffers) in situ and modulate inter- and
intra-molecular interactions.
This thesis reports the application of a single molecule technique, dual beam optical
tweezers, for the study of single biomolecules. A range of single molecule systems
was investigated such as i)VirE2 protein DNA machinery, ii) DNA-surfactant, EtBr
(ethidium bromide), SYBRÂź Green-DNA interactions and iii) dsDNA denaturation
studies. In addition the development of the present experimental setup is described to
enable combined force measurement as well as single molecule fluorescence studies.
The presented biomolecular results provide new and complementary information on
the different biological systems demonstrating the diversity of experiments that can be
performed on single DNA molecules using optical tweezers.
Chapter one gives a brief introduction to optical tweezers, describes how optical
tweezers work, the physics behind it, details of the experimental setup and the method
of force calibration required in micromanipulation. Optical tweezers have opened
exciting avenues of research, especially in biology. Biologists will be able to
investigate the nature of molecular machines one by one, and infer from their
behavior those properties common to the population.
In chapter 2, we show how optical tweezers were employed to study the change in the
mechanical properties of single DNA molecules upon binding of small agents. The
first part of this chapter reports on the changes in mechanics of single dsDNA in the
presence of cationic and anionic surfactants (used as non-viral vectors in gene
therapy). The second part describes the interaction of DNA binding ligands (SYBRÂź
Green, EtBr) with individual DNA strands.
Agrobacterium tumefaciens (AT), a Gram-negative bacterium, evolved a complex and
unique mechanism to transfer a long single stranded DNA (ssDNA) molecule from its
cytoplasm to the eukaryotic host plant cell nucleus. Central to this mechanism,
chapter 3 discusses the results of the measurements on VirE2 protein interacting with
single stranded DNA (ssDNA). VirE2 protein is a multifunctional protein from AT
that coat the transferred-ssDNA (T-DNA), interacts with host factors assisting nuclear
import of the complex, forms channels in lipid bilayers and displays a highly
cooperative binding to ssDNA. The biological findings are presented in a new generic
model which can be used to explain how generation of forces helps bacterial DNA to
enter the plant cell based on our single molecule data.
Single molecule dsDNA denaturation, relevant in many molecular biological
experiments, induced by NaOH and mechanical pulling are studied in chapter 4. Here
optical tweezers experiments give access to the âmeltingâ of hydrogen bonds by
mechanical forces or alkali denaturation (NaOH) of dsDNA in real time. The
mechanical stability and the transition of dsDNA to ssDNA is investigated at different
ionic strength as well as in buffers. Fluorescent images of single λ DNA labeled with
SYBRÂź Green were observed up to forces â„ 65 pN and indicate a B-DNA to S âDNA
transition.
Chapter 5 describes the implementation of single-molecule fluorescence detection
(SMF) in optical tweezers. The design and instrumental capabilities of optical
tweezers combined with SMF are discussed in detail. The development of this
instrument provides a worldwide unique experimental setup and opens up new
possibilities in the studies of complex biological systems.
Finally chapter 6 summarizes the results of this thesis and discusses future
experimental applications. The appendices provide further details for DNA sample
preparation, molecular biology and chemical surface activation recipes, an instruction
manual for the setup and the list of currently published papers
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