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.

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.

Improving the signal-to-noise ratio of high-speed contact mode atomic force microscopy.

During high-speed contact mode atomic force microscopy, higher eigenmode flexural oscillations of the cantilever have been identified as the main source of noise in the resultant topography images. We show that by selectively filtering out the frequencies corresponding to these oscillations in the time domain prior to transforming the data into the spatial domain, significant improvements in image quality can be achieved.

Modelling oscillatory flexure modes of an atomic force microscope cantilever in contact mode whilst imaging at high speed.

Understanding the modal response of an atomic force microscope is important for the identification of image artefacts captured using contact-mode atomic force microscopy (AFM). As the scan rate of high speed AFM increases, these modes present themselves as ever clearer noise patterns as the frequency of cantilever vibration falls under the frequency of pixel collection. An Euler-Bernoulli beam equation is used to simulate the flexural modes of the cantilever of an atomic force microscope as it images a hard surface in contact mode. Theoretical results are compared with experimental recordings taken in the high speed regime, as well as previous analytical results. It is shown that the model can capture the mode shapes and resonance properties of the first four eigenmodes.

High-speed atomic force microscopy in slow motion--understanding cantilever behaviour at high scan velocities.

Using scanning laser Doppler vibrometer we have identified sources of noise in contact mode high-speed atomic force microscope images and the cantilever dynamics that cause them. By analysing reconstructed animations of the entire cantilever passing over various surfaces, we identified higher eigenmode oscillations along the cantilever as the cause of the image artefacts. We demonstrate that these can be removed by monitoring the displacement rather than deflection of the tip of the cantilever. We compare deflection and displacement detection methods whilst imaging a calibration grid at high speed and show the significant advantage of imaging using displacement.

Methods for imaging DNA in liquid with lateral molecular-force microscopy.

Shear force microscopy is not normally associated with the imaging of biomolecules in a liquid environment. Here we show that the recently developed scattered evanescent wave (SEW) detection system, combined with custom-designed vertically oriented cantilevers (VOCs), can reliably produce true non-contact images in liquid of DNA molecules. The range of cantilever spring constants for successful shear force imaging was experimentally identified between 0.05 and 0.09 N m(-1). Images of λ-DNA adsorbed on mica in distilled water were obtained at scan rates of 8000 pixels s(-1). A new constant-height force mapping mode for VOCs is also presented. This method is shown to control the vertical position of the tip in the sample plane with better than 1 nm accuracy. The force mode is demonstrated by mapping the shear force above λ-DNA molecules adsorbed on mica in a liquid environment at different tip-sample separations.

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.

Experimental observation of contact mode cantilever dynamics with nanosecond resolution.

We report the use of a laser Doppler vibrometer to measure the motion of an atomic force microscope contact mode cantilever during continuous line scans of a mica surface. With a sufficiently high density of measurement points the dynamics of the entire cantilever beam, from the apex to the base, can be reconstructed. We demonstrate nanosecond resolution of both rectangular and triangular cantilevers. This technique permits visualization and quantitative measurements of both the normal and lateral tip sample interactions for the first and higher order eigenmodes. The ability to derive quantitative lateral force measurements is of interest to the field of microtribology/nanotribology while the comprehensive understanding of the cantilever's dynamics also aids new cantilever designs and simulations.

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.

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.

Real-time nanofabrication with high-speed atomic force microscopy.

The ability to follow nanoscale processes in real-time has obvious benefits for the future of material science. In particular, the ability to evaluate the success of fabrication processes in situ would be an advantage for many in the semiconductor industry. We report on the application of a previously described high-speed atomic force microscope (AFM) for nanofabrication. The specific fabrication method presented here concerns the modification of a silicon surface by locally oxidizing the region in the vicinity of the AFM tip. Oxide features were fabricated during imaging, with relative tip-sample velocities of up to 10 cm s(-1), and with a data capture rate of 15 fps.

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.

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.

Pushing the boundaries of local oxidation nanolithography: short timescales and high speeds.

Fabrication of nanometre-scale structures in short timescales and with high throughput has great importance in the future of nanoscale science and technology. We show that the local oxidation of hydrogen-passivated silicon surfaces by intermittent-contact mode atomic force microscopy can be applied on timescales as low as 500 ns to create single oxide nanostructures with dimensions of 0.6 x 15 nm(2). Furthermore, we report on preliminary experiments demonstrating that local oxidation can also be achieved with relative tip-sample speeds in excess of 2 cm s(-1) in order to pattern larger areas. This was realised using a high-speed scan stage based on a quartz crystal resonator operating at 20 kHz.

A new detection system for extremely small vertically mounted cantilevers.

Detection techniques currently used in scanning force microscopy impose limitations on the geometrical dimensions of the probes and, as a consequence, on their force sensitivity and temporal response. A new technique, based on scattered evanescent electromagnetic waves (SEW), is presented here that can detect the displacement of the extreme end of a vertically mounted cantilever. The resolution of this method is tested using different cantilever sizes and a theoretical model is developed to maximize the detection sensitivity. The applications presented here clearly show that the SEW detection system enables the use of force sensors with sub-micron size, opening new possibilities in the investigation of biomolecular systems and high speed imaging. Two types of cantilevers were successfully tested: a high force sensitivity lever with a spring constant of 0.17 pN nm(-1) and a resonant frequency of 32 kHz; and a high speed lever with a spring constant of 50 pN nm(-1) and a resonant frequency of 1.8 MHz. Both these force sensors were fabricated by modifying commercial microcantilevers in a focused ion beam system. It is important to emphasize that these modified cantilevers could not be detected by the conventional optical detection system used in commercial atomic force microscopes.

High-speed AFM of human chromosomes in liquid.

Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

In situ annealing and thickening of single crystals of C294H590 observed by atomic force microscopy.

The annealing behavior of twice-folded crystals of the long-chain alkane C294H590 is examined in situ, in real time, by atomic force microscopy (AFM). AFM is capable of following processes in real time provided that the time scale is sufficiently long for several images to be collected during the process. In this paper, we focus on the temperature dependence and the thickened morphology. We are able to investigate where the thickening starts and how this depends on temperature and how melting is influenced by morphology. By following the motion of holes within the crystal, a lower limit for the rate of diffusion of crystalline polyethylene is estimated. We also focus on the substrate effect on the crystal morphology and thickening, using mica, glass, and graphite.

Scanning probe evolution in biology.

Twenty years ago the first scanning probe instrument, the scanning tunneling microscope, opened up new realms for our perception of the world. Atoms that had been abstract entities were now real objects, clearly seen as distinguishable individuals at particular positions in space. A whole family of scanning probe instruments has been developed, extending our sense of touching to the scale of atoms and molecules. Such instruments are especially useful for imaging of biomolecular structures because they can produce topographic images with submolecular resolution in aqueous environments. Instruments with increased imaging rates, lower probe-specimen force interactions, and probe configurations not constrained to planar surfaces are being developed, with the goal of imaging processes at the single-molecule level-not only at surfaces but also within three-dimensional volumes-in real time.