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.

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.

Acoustic time-frequency domain imaging.

The axial resolution of conventional acoustic micro imaging is limited by the wavelength of acoustic waves. Acoustic time-frequency domain imaging was recently proposed to overcome the wavelength limit [Zhang et al., J. Acoust. Soc. Am. 118, 3706-3720 (2005)]. A continuous wavelet transform based acoustic time-frequency domain imaging technique is investigated in this paper. Experiments are performed on real 3D data collected from microelectronic packages. Results demonstrate the proposed technique reveals more image details and enhances the image contrast in comparison with conventional time domain imaging.

Clustering-based robust three-dimensional phase unwrapping algorithm.

Relatively recent techniques that produce phase volumes have motivated the study of three-dimensional (3D) unwrapping algorithms that inherently incorporate the third dimension into the process. We propose a novel 3D unwrapping algorithm that can be considered to be a generalization of the minimum spanning tree (MST) approach. The technique combines characteristics of some of the most robust existing methods: it uses a quality map to guide the unwrapping process, a region growing mechanism to progressively unwrap the signal, and also cut surfaces to avoid error propagation. The approach has been evaluated in the context of noncontact measurement of dynamic objects, suggesting a better performance than MST-based approaches.

Hybrid robust and fast algorithm for three-dimensional phase unwrapping.

We present a hybrid three-dimensional (3D) unwrapping algorithm that combines the strengths of two other fast and robust existing techniques. In particular, a branch-cut surface algorithm and a path-following method have been integrated in a symbiotic way, still keeping execution times within a range that permits their use in real-time applications that need a relatively fast solution to the problem. First, branch-cut surfaces are calculated, disregarding partial residue loops that end at the boundary of the 3D phase volume. These partial loops are then used to define a quality for each image voxel. Finally, unwrapping proceeds along a path determined by a minimum spanning tree (MST). The MST is built according to the quality of the voxels and avoids crossing the branch-cut surfaces determined at the first step. The resulting technique shows a higher robustness than any of the two methods used in isolation. On the one hand, the 3D MST algorithm benefits from the branch-cut surfaces, which endows it with a higher robustness to noise and open-ended wraps. On the other hand, incorrectly placed surfaces due to open loops at the boundaries in the branch-cut surface approach disappear.

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.

Residue vector, an approach to branch-cut placement in phase unwrapping: theoretical study.

What we believe to be a novel technique of branch-cut placement in the phase unwrapping is proposed. This approach is based on what we named residue vector, which is generated by a residue in a wrapped phase map and has an orientation that points out toward the balancing residue of opposite polarity. The residue vector can be used to guide the manner in which branch cuts are placed in phase unwrapping. Also, residue vector can be used for the determination of the weighting values used in different existing phase unwrapping methods such as minimum cost flow and least squares. The theoretical foundations of the residue-vector method are presented, and a branch-cut method using its information is developed and implemented. A general comparison is made between the residue-vector map and other existing quality maps.

Two-dimensional phase unwrapping using a hybrid genetic algorithm.

A novel hybrid genetic algorithm (HGA) is proposed to solve the branch-cut phase unwrapping problem. It employs both local and global search methods. The local search is implemented by using the nearest-neighbor method, whereas the global search is performed by using the genetic algorithm. The branch-cut phase unwrapping problem [a nondeterministic polynomial (NP-hard) problem] is implemented in a similar way to the traveling-salesman problem, a very-well-known combinational optimization problem with profound research and applications. The performance of the proposed algorithm was tested on both simulated and real wrapped phase maps. The HGA is found to be robust and fast compared with three well-known branch-cut phase unwrapping algorithms.

Spatial carrier fringe pattern demodulation by use of a two-dimensional continuous wavelet transform.

A novel technique that uses a fan two-dimensional (2D) continuous wavelet transform (CWT) to phase demodulate fringe patterns is proposed. The fan 2D CWT algorithm is tested by using computer generated and real fringe patterns. The result of this investigation reveals that the 2D CWT technique is capable of successfully demodulating fringe patterns. The proposed algorithm demodulates fringe patterns without the requirement of removing their background illumination prior to the demodulation process. Also, the algorithm is exceptionally robust against speckle noise. The performance of the 2D CWT technique in fringe pattern demodulation is compared with that of the 1D CWT algorithms. This comparison indicates that the 2D CWT outperforms its 1D counterpart for this application.

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.

Three-dimensional shape measurement of non-full-field reflective surfaces.

We describe a technique for the measurement of non-full-field reflective surfaces by using phase-stepping profilometry. We explain the principles of phase demodulation and discuss three-dimensional (3-D) height reconstruction in the case of measuring surfaces with reflective properties such as plain glass and mirrored glass. A number of required calibration algorithms are described to obtain surface slopes and reconstructed 3-D heights of the whole surface. Masking for non-full-field objects and the surface reconstruction procedure are demonstrated mathematically and algorithmically. Several experimental results are given for glass with different shapes and defects. Measurement of a spherical mirror with a known radius has also allowed us to show the performance of the proposed technique. This allows for the possibility to compare 3-D data from the known object with data taken from the measurement system.

Agglomerative clustering-based approach for two-dimensional phase unwrapping.

We describe a novel algorithm for two-dimensional phase unwrapping. The technique combines the principles of agglomerative clustering and use of heuristics to construct a discontinuous quality-guided path. Unlike other quality-guided algorithms, which establish the path at the start of the unwrapping process, our technique constructs the path as the unwrapping process evolves. This makes the technique less prone to error propagation, although it presents higher execution times than other existing algorithms. The algorithm reacts satisfactorily to random noise and breaks in the phase distribution. A variation of the algorithm is also presented that considerably reduces the execution time without affecting the results significantly.

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.

Fast two-dimensional phase-unwrapping algorithm based on sorting by reliability following a noncontinuous path.

We describe what is to our knowledge a novel technique for phase unwrapping. Several algorithms based on unwrapping the most-reliable pixels first have been proposed. These were restricted to continuous paths and were subject to difficulties in defining a starting pixel. The technique described here uses a different type of reliability function and does not follow a continuous path to perform the unwrapping operation. The technique is explained in detail and illustrated with a number of examples.

Robust, fast, and effective two-dimensional automatic phase unwrapping algorithm based on image decomposition.

We describe what is to our knowledge a novel approach to phase unwrapping. Using the principle of unwrapping following areas with similar phase values (homogenous areas), the algorithm reacts satisfactorily to random noise and breaks in the wrap distributions. Execution times for a 512 x 512 pixel phase distribution are in the order of a half second on a desktop computer. The precise value depends upon the particular image under analysis. Two inherent parameters allow tuning of the algorithm to images of different quality and nature.

Technique for phase measurement and surface reconstruction by use of colored structured light.

We present a new method for improving the measurement of three-dimensional (3-D) shapes by using color information of the measured scene as an additional parameter. The widest used algorithms for 3-D surface measurement by use of structured fringe patterns are phase stepping and Fourier fringe analysis. There are a number of problems and limitations inherent in these algorithms that include: that the phase maps produced are wrapped modulo 2pi, that in some cases the acquired fringe pattern does not fill the field of view, that there may be spatially isolated areas, and that there is often invalid and/or noisy data. The new method presented to our knowledge for the first time here uses multiple colored fringe patterns, which are projected at different angles onto the measured scene. These patterns are analyzed with a specially adapted multicolor version of the standard Fourier fringe analysis method. In this way a number of the standard difficulties outlined above are addressed.