Measuring nanoscale forces with living probes.

Optical trapping techniques have been used to investigate fundamental biological processes ranging from the identification of the processive mechanisms of kinesin and myosin to understanding the mechanics of DNA. To date, these investigations have relied almost exclusively on the use of isotropic probes based on colloidal microspheres. However, there are many potential advantages in utilizing more complex probe morphologies: use of multiple trapping points enables control of the interaction volume; increasing the distance between the optical trap and the sample minimizes photodamage in sensitive biological materials; and geometric anisotropy introduces the potential for asymmetric surface chemistry and multifunctional probes. Here we demonstrate that living cells of the freshwater diatom Nitzschia subacicularis Hustedt can be exploited as advanced probes for holographic optical tweezing applications. We characterize the optical and material properties associated with the high shape anisotropy of the silica frustule, examine the trapping behavior of the living algal cells, and demonstrate how the diatoms can be calibrated for use as force sensors and as force probes in the presence of rat B-cell hybridoma (11B11) cells.

An optically actuated surface scanning probe.

We demonstrate the use of an extended, optically trapped probe that is capable of imaging surface topography with nanometre precision, whilst applying ultra-low, femto-Newton sized forces. This degree of precision and sensitivity is acquired through three distinct strategies. First, the probe itself is shaped in such a way as to soften the trap along the sensing axis and stiffen it in transverse directions. Next, these characteristics are enhanced by selectively position clamping independent motions of the probe. Finally, force clamping is used to refine the surface contact response. Detailed analyses are presented for each of these mechanisms. To test our sensor, we scan it laterally over a calibration sample consisting of a series of graduated steps, and demonstrate a height resolution of ∼ 11 nm. Using equipartition theory, we estimate that an average force of only ∼ 140 fN is exerted on the sample during the scan, making this technique ideal for the investigation of delicate biological samples.

Position clamping of optically trapped microscopic non-spherical probes.

We investigate the degree of control that can be exercised over an optically trapped microscopic non-spherical force probe. By position clamping translational and rotational modes in different ways, we are able to dramatically improve the position resolution of our probe with no reduction in sensitivity. We also demonstrate control over rotational-translational coupling, and exhibit a mechanism whereby the average centre of rotation of the probe can be displaced away from its centre.

Surface imaging using holographic optical tweezers.

We present an imaging technique using an optically trapped cigar-shaped probe controlled using holographic optical tweezers. The probe is raster scanned over a surface, allowing an image to be taken in a manner analogous to scanning probe microscopy (SPM), with automatic closed loop feedback control provided by analysis of the probe position recorded using a high speed CMOS camera. The probe is held using two optical traps centred at least 10 µm from the ends, minimizing laser illumination of the tip, so reducing the chance of optical damage to delicate samples. The technique imparts less force on samples than contact SPM techniques, and allows highly curved and strongly scattering samples to be imaged, which present difficulties for imaging using photonic force microscopy. To calibrate our technique, we first image a known sample--the interface between two 8 µm polystyrene beads. We then demonstrate the advantages of this technique by imaging the surface of the soft alga Pseudopediastrum. The scattering force of our laser applied directly onto this sample is enough to remove it from the surface, but we can use our technique to image the algal surface with minimal disruption while it is alive, not adhered and in physiological conditions. The resolution is currently equivalent to confocal microscopy, but as our technique is not diffraction limited, there is scope for significant improvement by reducing the tip diameter and limiting the thermal motion of the probe.

Calibration of optically trapped nanotools.

Holographically trapped nanotools can be used in a novel form of force microscopy. By measuring the displacement of the tool in the optical traps, the contact force experienced by the probe can be inferred. In the following paper we experimentally demonstrate the calibration of such a device and show that its behaviour is independent of small changes in the relative position of the optical traps. Furthermore, we explore more general aspects of the thermal motion of the tool.

Hands-on with optical tweezers: a multitouch interface for holographic optical trapping.

We report the implementation of a multitouch console for control of a holographic optical tweezers system. This innovative interface enables the independent but simultaneous interactive control of numerous optical traps by multiple users, overcoming the limitations of traditional interfaces and placing the full power of holographic optical tweezing into the operators' hands.

Mapping real-time images of high-speed AFM using multitouch control.

Conventional AFM is highly restricted by its scan rate, a problem that has been overcome by the development of high-speed AFM systems. As the technology to produce higher scan rates has developed it has pushed forward the design of control software. However, the user interface has not evolved at the same rate, limiting the user to sequential control steps. In this paper we demonstrate the integration of HSAFM with a multitouch interface to produce a highly intuitive and responsive control environment. This enables nanometre resolution to be maintained whilst scanning the sample over tens of microns, and arbitrary paths to be traversed. We illustrate this by scanning around two chromosomes in water, before scanning on top of the chromosome, showing the surface structure.

Constructing 3D crystal templates for photonic band gap materials using holographic optical tweezers.

A simple and robust method is presented for the construction of 3-dimensional crystals from silica and polystyrene microspheres. The crystals are suitable for use as templates in the production of three-dimensional photonic band gap (PBG) materials. Manipulation of the microspheres was achieved using a dynamic holographic assembler (DHA) consisting of computer controlled holographic optical tweezers. Attachment of the microspheres was achieved by adjusting their colloidal interactions during assembly. The method is demonstrated by constructing a variety of 3-dimensional crystals using spheres ranging in size from 3 microm down to 800 nm. A major advantage of the technique is that it may be used to build structures that cannot be made using self-assembly. This is illustrated through the construction of crystals in which line defects have been deliberately included, and by building simple cubic structures.

Experimental study of the fluctuation theorem in a nonequilibrium steady state.

The fluctuation theorem (FT) quantifies the probability of second law violations in small systems over short time scales. While this theorem has been experimentally demonstrated for systems that are perturbed from an initial equilibrium state, there are a number of studies suggesting that the theorem applies asymptotically in the long time limit to systems in a nonequilibrium steady state. The asymptotic application of the FT to such nonequilibrium steady states has been referred to in the literature as the steady-state fluctuation theorem (or SSFT). In this paper, we demonstrate experimentally the application of the FT to nonequilibrium steady states, using a colloidal particle localized in a translating optical trap. Furthermore, we show, for this colloidal system, that the FT holds under nonequilibrium steady states for all time, and not just in the long time limit, as in the SSFT.

Reversibility in nonequilibrium trajectories of an optically trapped particle.

The fluctuation theorem (FT) describes how a system's thermodynamic irreversibility develops in time from a completely thermodynamically reversible system at short observation times, to a thermodynamically irreversible one at infinitely long times. In this paper, we present a general definition of the dissipation function Omega(t), the quantitative argument in the fluctuation theorem (FT), that is a measure of a system's irreversibility. Originally cast for deterministic systems, we demonstrate, through the example of two recent experiments, that the dissipation function can be defined for stochastic systems. While the ensemble average of Omega(t) is positive definite irrespective of the system for which it is constructed, different expressions for Omega(t) can arise in stochastic and deterministic systems. Moreover, within the stochastic framework, Omega(t) is not unique. Nevertheless, each of these expressions for Omega(t) satisfies the FT.

A compact holographic optical tweezers instrument.

Holographic optical tweezers have found many applications including the construction of complex micron-scale 3D structures and the control of tools and probes for position, force, and viscosity measurement. We have developed a compact, stable, holographic optical tweezers instrument which can be easily transported and is compatible with a wide range of microscopy techniques, making it a valuable tool for collaborative research. The instrument measures approximately 30×30×35 cm and is designed around a custom inverted microscope, incorporating a fibre laser operating at 1070 nm. We designed the control software to be easily accessible for the non-specialist, and have further improved its ease of use with a multi-touch iPad interface. A high-speed camera allows multiple trapped objects to be tracked simultaneously. We demonstrate that the compact instrument is stable to 0.5 nm for a 10 s measurement time by plotting the Allan variance of the measured position of a trapped 2 µm silica bead. We also present a range of objects that have been successfully manipulated.

The Kawasaki identity and the Fluctuation Theorem.

In this paper we show that the Fluctuation Theorem of Evans and Searles [D. J. Evans, D. J. Searles, Phys. Rev. E 50, 1645 (1994)] implies that the Kawasaki function exp(-Omega(t)) is unity for all time t. We confirm this relationship using experimental data obtained using optical tweezers, and show that the Kawasaki function is a valuable diagnostic tool.

Fluctuations and irreversibility: an experimental demonstration of a second-law-like theorem using a colloidal particle held in an optical trap.

The puzzle of how time-reversible microscopic equations of mechanics lead to the time-irreversible macroscopic equations of thermodynamics has been a paradox since the days of Boltzmann. Boltzmann simply sidestepped this enigma by stating "as soon as one looks at bodies of such small dimension that they contain only very few molecules, the validity of this theorem [the second law of thermodynamics and its description of irreversibility] must cease." Today we can state that the transient fluctuation theorem (TFT) of Evans and Searles is a generalized, second-law-like theorem that bridges the microscopic and macroscopic domains and links the time-reversible and irreversible descriptions. We apply this theorem to a colloidal particle in an optical trap. For the first time, we demonstrate the TFT in an experiment and show quantitative agreement with Langevin dynamics.