The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour.

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods.

The effects of acoustic vibration on fibroblast cell migration.

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (µm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration.

Analysis of dynamic cantilever behavior in tapping mode atomic force microscopy.

Tapping mode atomic force microscopy (AFM) provides phase images in addition to height and amplitude images. Although the behavior of tapping mode AFM has been investigated using mathematical modeling, comprehensive understanding of the behavior of tapping mode AFM still poses a significant challenge to the AFM community, involving issues such as the correct interpretation of the phase images. In this paper, the cantilever's dynamic behavior in tapping mode AFM is studied through a three dimensional finite element method. The cantilever's dynamic displacement responses are firstly obtained via simulation under different tip-sample separations, and for different tip-sample interaction forces, such as elastic force, adhesion force, viscosity force, and the van der Waals force, which correspond to the cantilever's action upon various different representative computer-generated test samples. Simulated results show that the dynamic cantilever displacement response can be divided into three zones: a free vibration zone, a transition zone, and a contact vibration zone. Phase trajectory, phase shift, transition time, pseudo stable amplitude, and frequency changes are then analyzed from the dynamic displacement responses that are obtained. Finally, experiments are carried out on a real AFM system to support the findings of the simulations.

Aiding phase unwrapping by increasing the number of residues in two-dimensional wrapped-phase distributions.

In phase unwrapping residues are points of locally inconsistent phase that occur within a wrapped-phase map, which are usually regarded as being problematic for phase-unwrapping algorithms. Real phase maps typically contain a number of residues that are approximately proportional to the subsequent difficulty in unwrapping the phase distribution. This paper suggests the radical use of the discrete Fourier transform to actually increase the number of residues in 2D phase-wrapped images that contain discontinuities. Many of the additional residues that are artificially generated by this method are located on these discontinuities. For example, in fringe projection systems, such phase discontinuities may come from physical discontinuity between different parts of the object, or by shadows cast by the object. The suggested technique can improve the performance of path independent phase-unwrapping algorithms because these extra residues simplify the process of setting the branch cuts in the wrapped image based on the distance to the nearest residue. The generated residues can also be used to construct more reliable quality maps and masks. The paper includes an initial analysis upon simulated phase maps and goes on to verify the results on a real experimental wrapped-phase distribution.

Absolute distance measurement with micrometer accuracy using a Michelson interferometer and the iterative synthetic wavelength principle.

We present a novel system that can measure absolute distances of up to 300 mm with an uncertainty of the order of one micrometer, within a timeframe of 40 seconds. The proposed system uses a Michelson interferometer, a tunable laser, a wavelength meter and a computer for analysis. The principle of synthetic wave creation is used in a novel way in that the system employs an initial low precision estimate of the distance, obtained using a triangulation, or time-of-flight, laser system, or similar, and then iterates through a sequence of progressively smaller synthetic wavelengths until it reaches micrometer uncertainties in the determination of the distance. A further novel feature of the system is its use of Fourier transform phase analysis techniques to achieve sub-wavelength accuracy. This method has the major advantages of being relatively simple to realize, offering demonstrated high relative precisions better than 5 × 10(-5). Finally, the fact that this device does not require a continuous line-of-sight to the target as is the case with other configurations offers significant advantages.

Actin bundling and polymerisation properties of eukaryotic elongation factor 1 alpha (eEF1A), histone H2A-H2B and lysozyme in vitro.

Elongation factor 1 alpha (eEF1A) is a positively charged protein which has been shown to interact with the actin cytoskeleton. However, to date, a specific actin binding site within the eEF1A sequence has not been identified and the mechanism by which eEF1A interacts with actin remains unresolved. Many protein-protein interactions occur as a consequence of their physicochemical properties and actin bundle formation has been shown to result from non-specific electrostatic interaction with basic proteins. This study investigated interactions between actin, eEF1A and two other positively charged proteins which are not regarded as classic actin binding proteins (namely lysozyme and H2A-H2B) in order to compare their actin organising effects in vitro. For the first time using atomic force microscopy (AFM) we have been able to image the interaction of eEF1A with actin and the subsequent bundling of actin in vitro. Interestingly, we found that eEF1A dramatically increases the rate of polymerisation (45-fold above control levels). We also show for the first time that H2A-H2B has remarkably similar effects upon actin bundling (relative bundle size/number) and polymerisation (35-fold increase above control levels) as eEF1a. The presence of lysozyme resulted in bundles which were distinct from those formed due to eEF1A and H2A-H2B. Lysozyme also increased the rate of actin polymerisation above the control level (by 10-fold). Given the striking similarities between the actin bundling and polymerisation properties of eEF1A and H2A-H2B, our results hint that dimerisation and electrostatic binding may provide clues to the mechanism through which eEF1A-actin bundling occurs.

Evaluation and benchmarking of a pixel-shifting camera for superresolution lensless digital holography.

In lensless digital holography, the comparatively low resolution of the CCD devices that are used to record the digital holograms has to date limited both the maximum linear dimensions of the measurement object and also the minimum possible stand-off distance between the object and the CCD detector. A signal-processing-based technique known as superresolution (SR) image reconstruction can provide an alternative approach that reduces these restrictions. We report on an SR image reconstruction technique that has been introduced by employing a camera with a "microscanning" function to capture SR digital holograms via multiple subpixel movements of the CCD sensor. A detailed description of the approach is given, along with experimental results, which are discussed and evaluated, showing the advantages of using this method. An approach using three-dimensional holographic contouring is also described that may be adopted as a strategy for benchmarking newly developed algorithms at any stage of the lensless digital holographic process.

Dynamic recalibration of scalable fringe-projection systems for large-scale object metrology.

Three-dimensional (3D) surface shape measurement is a vital component in many industrial processes. The subject has developed significantly over recent years and a number of mainly noncontact techniques now exist for surface measurement, exhibiting varying levels of maturity. Within the larger group of 3D measurement techniques, one of the most promising approaches is provided by those methods that are based upon fringe analysis. Current techniques mainly focus on the measurement of small and medium-scale objects, while work on the measurement of larger objects is not so well developed. One potential solution for the measurement of large objects that has been proposed by various researchers is the concept of performing multipanel measurement and the system proposed here uses this basic approach, but in a flexible form of a single moveable sensor head that would be cost effective for measuring very large objects. Most practical surface measurement techniques require the inclusion of a calibration stage to ensure accurate measurements. In the case of fringe analysis techniques, phase-to-height calibration is required, which includes the use of phase-to-height models. Most existing models (both analytical and empirical) are intended to be used in a static measurement mode, which means that, typically, a single calibration is performed prior to multiple measurements being made using an unvarying system geometry. However, multipanel measurement strategies do not necessarily keep the measurement system geometry constant and thus require dynamic recalibration. To solve the problem of dynamic recalibration, we propose a class of models called hybrid models. These hybrid models inherit the basic form of analytical models, but their coefficients are obtained in an empirical manner. The paper also discusses issues associated with all phase-to-height models used in fringe analysis that have a quotient form, identifying points of uncertainty and regions of distortion as issues affecting accuracy in phase maps produced in this manner.

Three-dimensional phase unwrapping using the Hungarian algorithm.

We propose a three-dimensional phase unwrapping technique that uses the Hungarian algorithm to join together all the partial residual loops that may occur in a wrapped phase volume. Experimental results have shown that the proposed algorithm is more robust and reliable than other well-known three-dimensional phase unwrapping algorithms. Additionally, the proposed algorithm is fast in terms of computational complexity, which makes it suitable for practical applications.

Robust three-dimensional best-path phase-unwrapping algorithm that avoids singularity loops.

In this paper we propose a novel hybrid three-dimensional phase-unwrapping algorithm, which we refer to here as the three-dimensional best-path avoiding singularity loops (3DBPASL) algorithm. This algorithm combines the advantages and avoids the drawbacks of two well-known 3D phase-unwrapping algorithms, namely, the 3D phase-unwrapping noise-immune technique and the 3D phase-unwrapping best-path technique. The hybrid technique presented here is more robust than its predecessors since it not only follows a discrete unwrapping path depending on a 3D quality map, but it also avoids any singularity loops that may occur in the unwrapping path. Simulation and experimental results have shown that the proposed algorithm outperforms its parent techniques in terms of reliability and robustness.

Fast and robust three-dimensional best path phase unwrapping algorithm.

What we believe to be a novel three-dimensional (3D) phase unwrapping algorithm is proposed to unwrap 3D wrapped-phase volumes. It depends on a quality map to unwrap the most reliable voxels first and the least reliable voxels last. The technique follows a discrete unwrapping path to perform the unwrapping process. The performance of this technique was tested on both simulated and real wrapped-phase maps. And it is found to be robust and fast compared with other 3D phase unwrapping algorithms.

Spatial fringe pattern analysis using the two-dimensional continuous wavelet transform employing a cost function.

We present a novel ridge extraction algorithm for use with the two-dimensional continuous wavelet transform to extract the phase information from a fringe pattern. A cost function is employed for the detection of the ridge. The results of the proposed algorithm on simulated and real fringe patterns are illustrated. Moreover, the proposed algorithm outperforms the maximum ridge extraction algorithm and it is found to be robust and reliable.

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy.

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.

Quantization error of CCD cameras and their influence on phase calculation in fringe pattern analysis.

We present an investigation into the phase errors that occur in fringe pattern analysis that are caused by quantization effects. When acquisition devices with a limited value of camera bit depth are used, there are a limited number of quantization levels available to record the signal. This may adversely affect the recorded signal and adds a potential source of instrumental error to the measurement system. Quantization effects also determine the accuracy that may be achieved by acquisition devices in a measurement system. We used the Fourier fringe analysis measurement technique. However, the principles can be applied equally well for other phase measuring techniques to yield a phase error distribution that is caused by the camera bit depth.

Opto-electronic sensing of body surface topology changes during radiotherapy for rectal cancer.

PURPOSE: The CT body surface underpins millimeter scale dose computation in radical radiotherapy. A lack of technology has prevented measurement of surface topology changes during irradiation. Consequently, body changes are incorporated into plans statistically. We describe the technology for dynamic measurement of continuous surface topology at submillimeter resolution and suggest appropriately modified planning. MATERIALS AND METHODS: An interferometer casts cosinusoidal fringes across the surface of a patient on a treatment couch. Motion-induced changes to the spatial phase of the fringes are used to generate dynamic sequences of body height maps. Volume-conserving CT warping, guided by height change, is used to illustrate potential planning perturbations. RESULTS: We present the results for a prone patient with rectal carcinoma. At most of the simultaneously measured 440 x 440 points in each of the 898 body height maps in a dynamic sequence, the standard deviations were <1-2 mm, with occasional points of 6 mm. Surface motion predominantly occurred along the small of the back. This motion was periodic and could take the spine and bladder across the 95% isodose contour. CONCLUSIONS: Surface changes are most likely to be within 3 mm during irradiation, despite the effects of breathing and the discomfort of lying prone. The dosimetric effects are acceptable.