An observational pilot study to assess the potential of a microfluidic tissue culture model to predict rectal cancer response to neo-adjuvant therapy

Radiotherapy has been reported to induce apoptosis and prevent the proliferation of malignant cells. Complete clinical response to neo-adjuvant long course chemoradiotherapy has been identified in up to 30% of patients with locally advanced rectal cancer. The aim of this study was firstly to maintain rectal cancer biopsies in a viable state within a microfluidic device and subsequently interrogate this ex vivo rectal cancer tissue with radiation and measure changes in morphology and induction of cell death through apoptosis.Murine colorectal tissue was used for initial optimisation, followed by biopsies from patients with locally advanced rectal cancer taken prior to neo- adjuvant therapy. This tissue was maintained in a biomimetic environment within a bespoke, glass microfluidic device. Subsequently, murine tissue was interrogated with single fractions of radiation (2Gy, 10Gy or 30Gy) to identify suitable doses for delivery to human tissue. Morphology was assessed using H&E staining of the tissue. Effluent from the tissue was collected for subsequent analysis of cell death using a lactate dehydrogenase (LDH) assay and metabolite release using a mass spectrometry-metabolomics approach. Apoptosis was evaluated using the M30 CytoDeath™ monoclonal antibody and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay to identify DNA fragmentation.Tissue was successfully maintained for over 70 hours with evidence of viability, as determined by preservation of morphology and increased LDH release after lysis. Rectal cancer biopsies (n = 11 patients) were subsequently interrogated with radiation. Only high doses of radiation (30Gy) delivered to murine colorectal tissue reproducibly induced high levels of LDH release, however architectural losses were seen in all tissue after irradiation regardless of dose. Human tissue was therefore irradiated with 2Gy as an approximation of the dose delivered clinically.Levels of apoptosis using M30 CytoDeath™ ELISA were not significantly increased in the irradiated groups when compared to control groups. However, using immunohistochemical assessment with M30 CytoDeath™ and TUNEL, significant increases in the irradiated groups were seen (p < 0.05). Evaluation of individual patients using these markers identified several patients with significant rises (p < 0.05) in levels of apoptosis, however there was no correlation with clinical response. Metabolomic analysis identified 28 differentially expressed (p < 0.0001) compounds in effluents collected prior to and after irradiation, however this appeared to be a time-dependent effect, rather than due to irradiation. This work has demonstrated that the microfluidic device can be used to reliably maintain both ex vivo healthy murine colorectal and human rectal cancer tissue for a sufficient period of time to permit interrogation with radiation. Findings demonstrated that apoptosis and morphological changes are induced by irradiation. Further work is required to correlate findings with clinical outcome, but important progress has been made to allow use of this platform as a predictive tool of response to neo-adjuvant therapy to deliver personalised therapy

Ultrastable heterodyne interferometry using a modulated light camera

Interferometry is used in a wide variety of fields for the instrumentation and analysis of subjects and the environment. When light beams interfere, an interference fringe pattern is generated. Captured widefield interference patterns can be used to determine changes in the optical path length of interfering beams across a 2D area. Two interferometer schemes regularly implemented in modern systems include the homodyne interferometer, where light with the same optical frequency in used to generate static intensity fringe patterns, and the heterodyne interferometer, where light with different optical frequencies are used to generate a fringe pattern that is modulated at a frequency equal to the optical frequency difference (beat frequency). A widefield heterodyne system is not straightforward to bring into practice, however, it does offer some benefits over a comparable homodyne interferometer, such as direct phase interpretation and the suppression of low frequency background light in interferograms. In this thesis, a widefield heterodyne interferometer system is presented. A custom prototype modulated light camera (MLC) chip was used to capture both homodyne and heterodyne fringe patterns. The 32x32 pixel camera is capable of continuously demodulating incident modulated light at frequencies between 100kHz and 17MHz. In the presented system, an error in the interferogram phase was determined to be Δφ = ±0.16radians (~9.1º). Comparisons between homodyne and heterodyne interferograms, captured using the MLC, are also presented. With modifications to the system, an ultrastable widefield heterodyne interferometer system was implemented. The intention of this system was to eliminate the contribution of piston phase to a captured interferogram without the need for common path optics. In contrast to the standard heterodyne setup, the reference signal used in the demodulation process was derived from one of the pixels on-board the MLC, rather than from an external source. This new local reference signal tracks the common changes in the temporal phase detected by all the MLC's pixels, eliminating piston phase and substantially reducing the contributions of unwanted vibrations and microphonics from interferograms. To demonstrate this ultrastable system, it is incorporated into a Mach-Zehnder interferometer, where a vibration is induced onto an object arm mirror (using a mounted speaker) at various frequencies. Stable interferograms are captured with the mirror moving at up to 85mm/s at 62Hz (an optical path length of 220μm, or 350 wavelengths for λ = 633nm), however, this limit was the result of the complex motion in the mirror mount rather than the stability limit of the system. The system is shown to be insensitive to pure piston phase variations equivalent to an object velocity of over 3m/s. As an application of the ultrastable system, a novel interferometer has been developed that captures the widefield fringe patterns generated by interfering two independent light sources, rather than by a single split source. The two separately stabilised HeNe lasers, constructed in the laboratory, produce light with a reasonably stable output frequency. Interfering two of these sources produce a heterodyne interference pattern with an unknown beat frequency. The beat frequency continuously varies because of the variation in the output frequency of each laser, but these stabilised lasers produce a beat frequency that drift by as little as 3MHz over 30 minutes. As the ultrastable system tracks changes in the temporal phase and instantaneous frequency of an incident fringe pattern, it can be used to track the variations in the modulation frequency generated by the fluctuations in the two separate lasers. The separation between the two lasers with regards to the images presented was about 35cm, but they can be separated by much larger amounts

Ultrastable heterodyne interferometry using a modulated light camera

Interferometry is used in a wide variety of fields for the instrumentation and analysis of subjects and the environment. When light beams interfere, an interference fringe pattern is generated. Captured widefield interference patterns can be used to determine changes in the optical path length of interfering beams across a 2D area. Two interferometer schemes regularly implemented in modern systems include the homodyne interferometer, where light with the same optical frequency in used to generate static intensity fringe patterns, and the heterodyne interferometer, where light with different optical frequencies are used to generate a fringe pattern that is modulated at a frequency equal to the optical frequency difference (beat frequency). A widefield heterodyne system is not straightforward to bring into practice, however, it does offer some benefits over a comparable homodyne interferometer, such as direct phase interpretation and the suppression of low frequency background light in interferograms. In this thesis, a widefield heterodyne interferometer system is presented. A custom prototype modulated light camera (MLC) chip was used to capture both homodyne and heterodyne fringe patterns. The 32x32 pixel camera is capable of continuously demodulating incident modulated light at frequencies between 100kHz and 17MHz. In the presented system, an error in the interferogram phase was determined to be Δφ = ±0.16radians (~9.1º). Comparisons between homodyne and heterodyne interferograms, captured using the MLC, are also presented. With modifications to the system, an ultrastable widefield heterodyne interferometer system was implemented. The intention of this system was to eliminate the contribution of piston phase to a captured interferogram without the need for common path optics. In contrast to the standard heterodyne setup, the reference signal used in the demodulation process was derived from one of the pixels on-board the MLC, rather than from an external source. This new local reference signal tracks the common changes in the temporal phase detected by all the MLC's pixels, eliminating piston phase and substantially reducing the contributions of unwanted vibrations and microphonics from interferograms. To demonstrate this ultrastable system, it is incorporated into a Mach-Zehnder interferometer, where a vibration is induced onto an object arm mirror (using a mounted speaker) at various frequencies. Stable interferograms are captured with the mirror moving at up to 85mm/s at 62Hz (an optical path length of 220μm, or 350 wavelengths for λ = 633nm), however, this limit was the result of the complex motion in the mirror mount rather than the stability limit of the system. The system is shown to be insensitive to pure piston phase variations equivalent to an object velocity of over 3m/s. As an application of the ultrastable system, a novel interferometer has been developed that captures the widefield fringe patterns generated by interfering two independent light sources, rather than by a single split source. The two separately stabilised HeNe lasers, constructed in the laboratory, produce light with a reasonably stable output frequency. Interfering two of these sources produce a heterodyne interference pattern with an unknown beat frequency. The beat frequency continuously varies because of the variation in the output frequency of each laser, but these stabilised lasers produce a beat frequency that drift by as little as 3MHz over 30 minutes. As the ultrastable system tracks changes in the temporal phase and instantaneous frequency of an incident fringe pattern, it can be used to track the variations in the modulation frequency generated by the fluctuations in the two separate lasers. The separation between the two lasers with regards to the images presented was about 35cm, but they can be separated by much larger amounts

SIMULTANEOUS RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF FENOFIBRATE AND ROSUVASTATIN CALCIUM IN BULK AND TABLET DOSAGE FORM

An isocratic reversed-phase liquid chromatograpic assay method was developed for the quantitative determination of fenofibrate and rosuvastatin calcium in bulk and tablet dosage form. A Lichrosphere Select-B C8 (250x4.6mm & 5.0μm) column with a mobile phase containing Solution A (Milli-Q water has pH3.0 made by orthophosphoric acid): Methanol (20:80). The flow rate was 1.0 mL min−1: 1.5 ml/min and the detection of fenofibrate and rosuvastatin calcium was carried out on absorbance detector at 254nm.The retention times was 12 min (rosuvastatin- 3.40, fenofibrate-7.75). A linear response r2 > 1.0 for fenofibrate in the range of 40- 300μg/ml and r2 > 0.9997 in the range of 2.8-21μg/ml for rosuvastatin calcium was observed. The proposed method was validated with respect to system suitability, specificity and selectivity, stability of analytical solutions linearity, accuracy, precision, and robustness. The method was successfully applied to the estimation of fenofibrate and rosuvastatin calcium in bulk and tablet dosage form. Keywords: RP-HPLC, fenofibrate, rosuvastatin calcium, Validatio

Simple method of measuring thicknesses of surface-hardened layers by laser ultrasonic technique

We proposed a simple non-contact inspection method using a laser ultrasonic technique to measure the thickness of a surface-hardened layer. This measurement is based on the dependence of a surface-wave velocity on the thickness of the hardened layers. In this case, it is essential to measure the surface-wave velocity with a wavelength comparable to the thickness of a hardened layer. However, it is not easy to selectively generate the surface waves with a desired wavelength. Thus we proposed the method where the surface waves with a desired wavelength are extracted using bandpass electrical filters. This method is simpler than the conventional one based on dispersion because the surface-wave velocity is directly obtained from measured temporal waveforms. We applied the method to measure the thickness of hardened layers and showed that the results were in good agreement with that obtained by the conventional method and numerical results

Orientation imaging of macro-sized polysilicon grains on wafers using spatially resolved acoustic spectroscopy

Due to its economical production process polysilicon, or multicrystalline silicon, is widely used to produce solar cell wafers. However, the conversion efficiencies are often lower than equivalent monocrystalline or thin film cells, with the structure and orientation of the silicon grains strongly linked to the efficiency. We present a non-destructive laser ultrasonic inspection technique, capable of characterising large (52 x 76 mm2) photocell's microstructure – measurement times, sample surface preparation and system upgrades for silicon scanning are discussed. This system, known as spatially resolved acoustic spectroscopy (SRAS) could be used to optimise the polysilicon wafer production process and potentially improve efficiency

Spatially resolved acoustic spectroscopy for selective laser melting

Additive manufacturing (AM) is a manufacturing technique that typically builds parts layer by layer, for example, in the case of selective laser melted (SLM) material by fusing layers of metal powder. This allows the construction of complex geometry parts, which, in some cases cannot be made by traditional manufacturing routes. Complex parts can be difficult to inspect for material conformity and defects which are limiting widespread adoption especially in high performance arenas. Spatially resolved acoustic spectroscopy (SRAS) is a technique for material characterisation based on robustly measuring the surface acoustic wave velocity. Here the SRAS technique is applied to prepare additively manufactured material to measure the material properties and identify defects. Results are presented tracking the increase in the measured velocity with the build power of the selective laser melting machine. Surface and subsurface defect measurements (to a depth of ∼24 μm) are compared to electron microscopy and X-ray computed tomography. It has been found that pore size remains the same for 140 W to 190 W melting power (mean: 115–119 μm optical and 134–137 μm velocity) but the number of pores increase significantly (70–126 optical, 95–182 velocity) with lower melting power, reducing overall material density

Widefield two laser interferometry

A novel system has been developed that can capture the wide- field interference pattern generated by interfering two independent and incoherent laser sources. The interferograms are captured using a custom CMOS modulated light camera (MLC) which is capable of demodulating light in the megahertz region. Two stabilised HeNe lasers were constructed in order to keep the optical frequency difference (beat frequency) between the beams within the operational range of the camera. This system is based on previously reported work of an ultrastable heterodyne interferometer [Opt. Express 20, 17722 (2012)]. The system used an electronic feedback system to mix down the heterodyne signal captured at each pixel on the camera to cancel out the effects of time varying piston phase changes observed across the array. In this paper, a similar technique is used to track and negate the effects of beat frequency variations across the two laser pattern. This technique makes it possible to capture the full field interferogram caused by interfering two independent lasers even though the beat frequency is effectively random. As a demonstration of the system’s widefield interferogram capture capability, an image of a phase shifting object is taken using a very simple two laser interferometer

Spatially resolved acoustic spectroscopy (SRAS) microstructural imaging

© 2019 Author(s). Spatially resolved acoustic spectroscopy (SRAS) is an acoustic microscopy technique that can image the microstructure and measure the crystallographic orientation of grains or crystals in the material. It works by measuring the velocity of surface acoustic waves (SAWs) via the acoustic spectrum. In the usual configuration, the SAWs are generated by laser using a pattern of lines and detected by laser at a point close to this grating-like source. The use of the acoustic spectrum as a means of measuring the velocity has a number of practical advantages which makes the technique robust and fast and gives good spatial resolution. This makes the measurement suitable for imaging and gives it many advantages over traditional laser UT and microstructural measurement techniques. As SRAS is a laser ultrasound testing technique (LUT) which can be applied to a wide range of industrially relevant samples as a non-destructive evaluation technique. There are no size limitations on the samples that can be imaged and the surface preparation required is significantly more relaxed than many other techniques with the capability of operating on many as manufactured finishes. This permits the use of SRAS as an online inspection tool, for instance during additive or subtractive manufacturing, as a QA tool during manufacture or as an NDE/T tool in service

Assessing the capability of in-situ nondestructive analysis during layer based additive manufacture

Unlike more established subtractive or constant volume manufacturing technologies, additive manufacturing methods suffer from a lack of in-situ monitoring methodologies which can provide informationrelating to process performance and the formation of defects. In-process evaluation for additive manufacturing is becoming increasingly important in order to assure the integrity of parts produced in this way. This paper addresses the generic performance of inspection methods suitable for additive manufacturing. Key process and measurement parameters are explored and the impacts these have upon production rates are defined. Essential working parameters are highlighted, within which the spatial opportunity and temporal penalty for measurement allow for comparison of the suitability of different nondestructive evaluation techniques. A new method of benchmarking in-situ inspection instruments and characterising their suitability for additive manufacturing processes is presented to act as a design tool to accommodate end user requirements. Two inspection examples are presented: spatially resolved acoustic spectroscopy and optical coherence tomography for scanning selective laser melting and selective laser sintering parts, respectively. Observations made from the analyses presented show that the spatial capability arising from scanning parameters affects the temporal penalty and hence impact upon production rates. A case study, created from simulated data, has been used to outline the spatial performance of a generic nondestructive evaluation method and to show how a decrease in data capture resolution reduces the accuracy of measurement