We show how measurements of forces through the analysis of light momentum changes can be combined with holographic optical tweezers (HOTs) to leverage the potential of this force detection method. As the magnitude is not derived from the sample displacement, no in situ calibration is required, and measurements are not restricted to specific conditions. In particular, we show that forces on irregular particles and beams can also be measured with optical traps by simultaneously applying a force in the same direction to multiple holographically-trapped particles through a constant flow. Finally, we measure forces exerted on micro-cylinders in order to assess their transversal and longitudinal drag coefficients.
In this work, we present and discuss several developments implemented in an instrument that uses the detection of the light momentum change for measuring forces in an optical trap. A system based on this principle provides a direct determination of this magnitude regardless of the positional response of the sample under the effect of an external force, and it is therefore to be preferred when in situ calibrations of the trap stiffness are not attainable or are difficult to achieve. The possibility to obtain this information without relying upon a harmonic model of the force is more general and can be used in a wider range of situations. Forces can be measured on non-spherical samples or non-Gaussian beams, on complex and changing environments, such as the interior of cells, or on samples with unknown properties (size, viscosity, etc.). However, the practical implementation of the method entails some difficulties due to the strict conditions in the design and operation of an instrument based on this method. We have focused on some particularly conflicting points. We developed a process and a mechanism to determine and systematically set the correct axial position of the device. We further analyzed and corrected the non-uniform transmittance of the optical system and we finally compensated for the variations in the sensor responsivity with temperature. With all these improvements, we obtained an accuracy of ~5% in force measurements for samples of different kinds.
The force exerted by optical tweezers can be measured by tracking the momentum changes of the trapping beam, a method which is more general and powerful than traditional calibration techniques as it is based on first principles, but which has not been brought to its full potential yet, probably due to practical difficulties when combined with high-NA optical traps, such as the necessity to capture a large fraction of the scattered light. We show that it is possible to measure forces on arbitrary biological objects inside cells without an in situ calibration, using this approach. The instrument can be calibrated by measuring three scaling parameters that are exclusively determined by the design of the system, thus obtaining a conversion factor from volts to piconewtons that is theoretically independent of the physical properties of the sample and its environment. We prove that this factor keeps valid inside cells as it shows good agreement with other calibration methods developed in recent years for viscoelastic media. Finally, we apply the method to measuring the stall forces of kinesin and dynein in living A549 cells.
We recently showed that force measurements through the detection of beam momentum changes can be implemented in
single-beam gradient traps. We thereby achieved a method that renders force measurements insensitive to sample's
geometrical and optical properties. Our instrument could be calibrated by a parameter that remained constant within a
4% error when, under comparable conditions, the traditional approach based on position detection could change by a
factor of two or more. The setup required for these measurements was but a modified version of the apparatus used for
position detection with back-focal-plane interferometry (BFPI). Despite the apparent difference between both techniques,
we show here that they are not independent and we explicitly indicate the connection between them. The results suggest
that our changes in the position detection instrument could have some important advantages and improve the technique
when this is used to ultimately determine optical forces.
Optical tweezers constitute a powerful technique with a wide range of applications in biological studies. Nevertheless,
the response of living matter to the interaction with laser light still remains unclear. Photothermal and photochemical
effects primarily due to laser absorption seem to be the major detrimental causes. This work aims at assessing the laser
heating effects on NG108 cells by studying the induction of apoptosis and necrosis on these cells. Furthermore,
quantification of the local temperature increase in the focus of the optical trap with the back-focal-plane interferometry
technique is also one of the main goals.
KEYWORDS: Lead, Proteins, Optical tweezers, Luminescence, Microscopy, Signal to noise ratio, In vitro testing, Video, Microscopes, Acquisition tracking and pointing
Zera® technology offers the possibility to artificially induce the formation of spherical organelles in different kinds of
cells. Their large size and high density, compared to the native organelles of the cell, make them good candidates to be
used as a handles for the realization of biophysical experiments inside living cells. Furthermore, they present a high
signal-to-noise ratio in fluorescence microscopy and small photobleaching. This work focuses mainly on the nature of
protein body motion in Nicotiana Benthamiana (tobacco) cells. The high-speed tracking of these structures reveals that
they move in a stepwise mode, suggesting that myosin XI motors directly pull these organelles through the cytosol. Our
results indicate that these artificially-induced structures are well integrated into the natural processes of the cell so that
the technique may be advantageous for the study of the intracellular transport mechanisms. Large forces can be exerted
with optical tweezers to mechanically interact with the moving organelles.
KEYWORDS: In vivo imaging, Optical tweezers, Calibration, Microscopes, Optical testing, Particles, Photodetectors, Sensors, In vitro testing, Signal detection
The stall forces of processive molecular motors have been widely studied previously in vitro. Even so, in vivo
experiments are required for determining the actual performance of each molecular motor in its natural environment. We
report the direct measurement of light momentum changes in single beam optical tweezers as a suitable technique for
measuring forces inside living cells, where few alternatives exist. The simplicity of this method, which does not require
force calibration for each trapped object, makes it convenient for measuring the forces involved in fast dynamic
biological processes such us intracellular traffic. Here we present some measurements of the stall force of processive
molecular motors inside living Allium cepa cells.
Optical tweezer experiments have partially unveiled the mechanical properties of processive motor proteins while driving polystyrene or silica microbeads in vitro. However, the set of forces underlying the more complex transport mechanisms in living samples remains poorly understood. Several studies have shown that optical tweezers are capable of trapping vesicles and organelles in the cytoplasm of living cells, which can be used as handles to mechanically interact with engaged (active) motors, or other components regulating transport. This may ultimately enable the exploration of the mechanics of this trafficking mechanism in vivo. These cell manipulation experiments have been carried out using different strategies to achieve dynamic beam steering capable of trapping these subcellular structures. We report here the first trapping and manipulation, to our knowledge, of such small motor-propelled cargos in living cells using holographic technology.
Digital holography enables the creation of multiple optical traps at arbitrary three-dimensional locations and spatial light modulators permit updating those holograms at video rates. However, the time required for computing the holograms makes interactive optical manipulation of several samples difficult to achieve. We introduce an algorithm for computing holographic optical tweezers that is both easy to implement and capable of speeds in excess of 10 Hz when running on a Pentium IV computer. A discussion of the pros and cons of the algorithm, a mathematical analysis of the efficiency of the resulting traps, as well as results of the three-dimensional manipulation of polystyrene micro spheres are included.
The paper describes the design of an inexpensive holographic optical tweezers setup. The setup is accompanied
by software that allows real-time manipulation of the sample and takes into account the experimental features of
the setup, such as aberration correction and LCD modulation. The LCD, a HoloEye LCR-2500, is the physical
support of the holograms, which are calculated using the fast random binary mask algorithm. The real-time
software achieves 12 fps at full LCD resolution (including aberration correction and modulation) when run on a
Pentium IV HT, 3.2 GHz computer.
We study how the aberrations of the optical system affect the correlation peak heights in a joint transform correlator. A Meopta objective is evaluated as Fourier-transform system, and the correlation results taking into account its aberrations are presented. Finally, a simple method to minimize the effect of the aberrations is proposed.
The operation of a joint transform correlator is based on two Fourier transforms in cascade. The first one produces the joint power spectrum; the second one yields the correlation. In this work, we study the influence of the aberrations that affect the optical systems that produce the two Fourier transforms, by computing the wave aberration of the diffraction setups from data obtained by ray tracing. First, we consider the aberration only in the power spectrum, then only in the transformation of power spectrum to give the correlation and finally, in the two steps at the same time. In the study, real conditions have been reproduced and the dimensions and resolutions of real liquid crystal-displays have been used. The results show that the quality of the correlation is determined mostly by the aberrations of the first diffraction stage.
Optical correlators process two-dimensional images that come from a three-dimensional world. Filters designed for object recognition of three-dimensional scenes must have the information of all possible views. This implies a large quantity of filters, especially when the object is moving with respect to the observer. Although filters designed through the synthetic discriminant functions formalism can encode information of several images, there is a practical limit imposed by the noise appearing at the correlation plane. Fast correlators are one way of solving this problem. In this work we propose a global process for detecting 3-D objects based on fast sequential correlations with filters derived from the different possible views of the target. The acquisition of these views is accomplished in a fast and simple way by means of a three-dimensional scanner based on stereovision techniques. The 3-D model of the object thus obtained is then used to compute synthetic plane views from any desired viewpoint. A compact correlator has been developed which uses fast CCD cameras for input and output, and ferroelectric SLMs (spatial light modulators) to display the scene and the sequence of filters. The process of digitizing the 3-D coordinates is described in detail, from the acquisition of the stereopair of images, the stereo-matching algorithm we use and the final integration of all data sets into a common object-centered coordinate system. Also, general engineering problems involved in the design and construction of the correlator are analysed and discussed.
This work presents a 3D scanner system based on stereovision techniques to generate plane views of an object from an arbitrary viewpoint. These views are used as the reference templates in an optical correlator system designed to recognize the object.
Fraunhofer diffraction at finite distances can be produced by a variety of optical setups. Convergent arrangements, in which the diffracting object is illuminated by a converging spherical wave-front, are of special interest since they enable an easy control over the size of the resulting diffraction pattern. In particular, such arrangements are frequently used in optical correlators as a means to obtain the Fourier transform in the input scene. We analyze the effect of the wave aberration introduced by the convergent optical system in the resulting Fourier transform, both experimentally and by a computer simulation, as well as its impact over the correlation plane. Also, we introduce a simple optimization method that gives good results in alleviating the loss of space invariance, which is the most important side effect found in the study.
Correlators, as any other sophisticated instrument, must be calibrated and adjusted for proper operation. We explore the necessity and feasibility of several techniques to tune its different elements, having in common that they do not require any external setup. This is accomplished by using the correlator to interrogate itself about the convenience of any modification of its nominal characteristics and thus it allows the correction of time-varying as well as correlator- dependent effects.
An implementation of a phase extractor Vander-Lugt correlator, which operates with a single spatial light modulator is suggested. Optical phase-retrieval manipulation, based on the symmetrization of the input scenes is proposed. As a consequence, the number of digital manipulations of the information is substantially reduced and spatial correlation filters can be used. Theoretical analysis and some simulated results are provided. The study also includes the results when the scenes are immersed into background or zero mean additive noise.
Phase-only filters are attractive designs because they provide good noise resistance and fairly narrow peaks together with optimum light efficiency. Since they contain only phase information they are easier to implement in current spatial light modulators. However, their extension to the synthetic discriminant function (SDF) formalism is troublesome owing to the nonlinear constraints involved. We developed a new procedure for computing phase-only SDF filters which is intended to overcome some of the pitfalls of previous methods.
The synthetic discriminant filter (SDF) is a linear combination of the target and the objects to be rejected. The coefficients of the combination are calculated in such a way that the central correlations with the different objects take predeterminate values. Nevertheless, The correlation values in other points are not controlled, and in consequence, high sidelobes may appear causing false alarms. In this communication we propose a method to solve this problem. The procedure is based on the use of two filters and the subsequent postprocessing of the correlation intensity distributions obtained.
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