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Publication Raman Spectroscopy: Twenty-five years of concept to clinical application for early diagnosis of pre-cancer in Barrett’s Oesophagus(Anita Publications, 2020-12-01) Day, John; Stone, Nick; Barr, Hugh; Kendall, Catherine; Hutchings, Joanne; Isabelle, Martin; Lloyd, Gavin; Almond, Mark; Pavlou, Alexandros; Upchurch, Emma; Dudgeon, Alexander; Noor Mohamed, Mohamed; Old, Oliver; Noor Mohamed, Mohamed; Dudgeon, Alexander; Old, Oliver; Pavlou, Alexandros; Lloyd, Gavin; Isabelle, Martin; Hutchings, Joanne; Kendall, Catherine; Barr, Hugh; Medical and DentalEarly diagnosis and treatment of all diseases and in particular cancer is important to allow curative treatment. Symptomatic cancer is usually a lethal disease that requires extensive treatment that is enormously challenging for the patient. Over some twenty-five years we have explored the use of Raman to detect molecular changes in precancerous change and cancer of the oesophagus. The aims have been to reduce the subjectivity of histological diagnosis in particular of dysplastic pre invasive cancerous changes in columnar lined (Barrett’s Oesophagus). These changes are often macroscopically invisible and very easily undetectable. Following this we have investigated the development of rapid diagnostic techniques to detect early degeneration in real time without the delays inherent in biopsy and histological analysis. In particular, we have concentrated on the early detection of the macroscopically invisible changes that precede the degeneration to cancer in some patients with Barrett’s Oesophagus. Once these changes are detected the area can we treated using endoscopic techniques and the progression to cancer interrupted without major and life threatening interventions.Publication The application of Raman spectroscopy to the diagnosis of mitochondrial muscle disease: A preliminary comparison between fibre optic probe and microscope formats(Wiley, 2021-11-02) Alix, James; Plesia, Maria; Lloyd, Gavin; Dudgeon, Alexander; Kendall, Catherine; McDermott, Christopher; Gorman, Grainne; Taylor, Robert; Shaw, Pamela; Day, John; Dudgeon, Alexander; Kendall, Catherine; Healthcare ScientistsMuscle biopsy remains an important component of the diagnostic repertoire for patients with suspected mitochondrial disease, underpinning specialist histopathological and biochemical analyses. Raman spectroscopy has not yet been applied to mitochondrial disease, and new fibre optic systems, with advantages in terms of cost and portability, could provide a rapid means to identify muscle pathology. In this study, we aimed to explore the potential of two different formats of Raman spectroscopy to identify mitochondrial disease: a miniaturised fibre optic Raman system and a standard commercial Raman microscope. Raman spectra were recorded from muscle samples from healthy volunteers (n = 10) and patients with genetically confirmed mitochondrial disease (n = 15). Multivariate classification algorithms demonstrated a high level of disease classification performance with both the fibre optic probe system and microscope (area under receiver operating characteristic curves 0.80–0.82). Key spectral changes associated with mitochondrial disease concerned the α-helical configuration of proteins. The results suggest that Raman spectroscopy of muscle is worthy of further investigation as a technique for the rapid identification of mitochondrial disease. Open ResearchPublication Rapid identification of human muscle disease with fibre optic Raman spectroscopy(Royal Society of Chemistry, 2022-04-21) Alix, James; Plesia, Maria; Lloyd, Gavin; Dudgeon, Alexander; Kendall, Catherine; Hewamadduma, Channa; Hadjivassiliou, Marios; McDermott, Christopher; Gorman, Grainne; Taylor, Robert; Shaw, Pamela; Day, John; Kendall, Catherine; Dudgeon, Alexander; Healthcare Scientistshe diagnosis of muscle disorders (“myopathies”) can be challenging and new biomarkers of disease are required to enhance clinical practice and research. Despite advances in areas such as imaging and genomic medicine, muscle biopsy remains an important but time-consuming investigation. Raman spectroscopy is a vibrational spectroscopy application that could provide a rapid analysis of muscle tissue, as it requires no sample preparation and is simple to perform. Here, we investigated the feasibility of using a miniaturised, portable fibre optic Raman system for the rapid identification of muscle disease. Samples were assessed from 27 patients with a final clinico-pathological diagnosis of a myopathy and 17 patients in whom investigations and clinical follow-up excluded myopathy. Multivariate classification techniques achieved accuracies ranging between 71–77%. To explore the potential of Raman spectroscopy to identify different myopathies, patients were subdivided into mitochondrial and non-mitochondrial myopathy groups. Classification accuracies were between 74–89%. Observed spectral changes were related to changes in protein structure. These data indicate fibre optic Raman spectroscopy is a promising technique for the rapid identification of muscle disease that could provide real time diagnostic information. The application of fibre optic Raman technology raises the prospect of in vivo bedside testing for muscle diseases which would significantly streamline the diagnostic pathway of these disorders.Publication Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study(American Chemical Society, 2020-11-21) Guo, Shuxia; Beleites, Claudia; Neugebauer, Ute; Abalde-Cela, Sara; Afseth, Nils; Alsamad, Fatima; Anand, Suresh; Araujo-Andrade, Cuauhtemoc; Aškrabic, Sonja; Avci, Ertug; Baia, Monica; Kendall, Catherine; Kendall, Catherine; Additional Professional Scientific and TechnicalThe variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a ‘primary’ setup and the test data are generated on ‘replicate’ setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.Publication Viscoelastic properties of biopolymer hydrogels determined by Brillouin spectroscopy: A probe of tissue micromechanics(American Association for the Advancement of Science, 2020-10-30) Bailey, Michelle; Alunni-Cardinali, Martina; Correa, Noemi; Caponi, Silvia; Holsgrove, Timothy; Barr, Hugh; Stone, Nick; Winlove, Peter; Fioretto, Daniele; Palombo, Francesca; Barr, Hugh; Medical and DentalMany problems in mechanobiology urgently require characterization of the micromechanical properties of cells and tissues. Brillouin light scattering has been proposed as an emerging optical elastography technique to meet this need. However, the information contained in the Brillouin spectrum is still a matter of debate because of fundamental problems in understanding the role of water in biomechanics and in relating the Brillouin data to low-frequency macroscopic mechanical parameters. Here, we investigate this question using gelatin as a model system in which the macroscopic physical properties can be manipulated to mimic all the relevant biological states of matter, ranging from the liquid to the gel and the glassy phase. We demonstrate that Brillouin spectroscopy is able to reveal both the elastic and viscous properties of biopolymers that are central to the structure and function of biological tissues.Publication In Vivo Fiber Optic Raman Spectroscopy of Muscle in Preclinical Models of Amyotrophic Lateral Sclerosis and Duchenne Muscular Dystrophy(American Chemical Society, 2021-05-05) Plesia, Maria; Stevens, Oliver; Lloyd, Gavin; Kendall, Catherine; Coldicott, Ian; Kennerley, Aneurin; Miller, Gaynor; Shaw, Pamela; Mead, Richard; Day, John; Alix, James; Lloyd, Gavin; Kendall, Catherine; Healthcare ScientistsNeuromuscular diseases result in muscle weakness, disability, and, in many instances, death. Preclinical models form the bedrock of research into these disorders, and the development of in vivo and potentially translational biomarkers for the accurate identification of disease is crucial. Spontaneous Raman spectroscopy can provide a rapid, label-free, and highly specific molecular fingerprint of tissue, making it an attractive potential biomarker. In this study, we have developed and tested an in vivo intramuscular fiber optic Raman technique in two mouse models of devastating human neuromuscular diseases, amyotrophic lateral sclerosis, and Duchenne muscular dystrophy (SOD1G93A and mdx, respectively). The method identified diseased and healthy muscle with high classification accuracies (area under the receiver operating characteristic curves (AUROC): 0.76-0.92). In addition, changes in diseased muscle over time were also identified (AUROCs 0.89-0.97). Key spectral changes related to proteins and the loss of α-helix protein structure. Importantly, in vivo recording did not cause functional motor impairment and only a limited, resolving tissue injury was seen on high-resolution magnetic resonance imaging. Lastly, we demonstrate that ex vivo muscle from human patients with these conditions produced similar spectra to those observed in mice. We conclude that spontaneous Raman spectroscopy of muscle shows promise as a translational research tool.Publication OGC P28 Raman Needle Probe Lymph Node Assessment for Oesophageal Cancer: The DOLOMITE Study(Oxford University Press, 2022-12-07) Ireland, Philip; Old, Oliver; Hornby, Steve; Kendall, Catherine; Barr, Hugh; Shore, Angela; Stone, Nick; Ireland, Philip; Old, Oliver; Hornby, Steve; Kendall, Catherine; Barr, Hugh; Medical and Dental; Healthcare ScientistsNo abstract availablePublication Fiber optic Raman spectroscopy for the evaluation of disease state in Duchenne muscular dystrophy: An assessment using the mdx model and human muscle(Wiley, 2022-07-15) Alix, James; Plesia, Maria; Hool, Sarah; Coldicott, Ian; Kendall, Catherine; Shaw Dbe, Pamela; Mead, Richard; Day, John; Kendall, Catherine; Healthcare ScientistsIntroduction/aims: Raman spectroscopy is an emerging technique for the evaluation of muscle disease. In this study we evaluate the ability of in vivo intramuscular Raman spectroscopy to detect the effects of voluntary running in the mdx model of Duchenne muscular dystrophy (DMD). We also compare mdx data with muscle spectra from human DMD patients. Methods: Thirty 90-day-old mdx mice were randomly allocated to an exercised group (48-hour access to a running wheel) and an unexercised group (n = 15 per group). In vivo Raman spectra were collected from both gastrocnemius muscles and histopathological assessment subsequently performed. Raman data were analyzed using principal component analysis-fed linear discriminant analysis (PCA-LDA). Exercised and unexercised mdx muscle spectra were compared with human DMD samples using cosine similarity. Results: Exercised mice ran an average of 6.5 km over 48 hours, which induced a significant increase in muscle necrosis (P = .03). PCA-LDA scores were significantly different between the exercised and unexercised groups (P < .0001) and correlated significantly with distance run (P = .01). Raman spectra from exercised mice more closely resembled human spectra than those from unexercised mice. Discussion: Raman spectroscopy provides a readout of the biochemical alterations in muscle in both the mdx mouse and human DMD muscle.Publication Non-negative matrix factorisation of Raman spectra finds common patterns relating to neuromuscular disease across differing equipment configurations, preclinical models and human tissue(Wiley, 2022-12-22) Alix, James; Plesia, Maria; Schooling, Chloe; Dudgeon, Alexander; Kendall, Catherine; Kadirkamanathan, Visakan; McDermott, Christopher; Gorman, Grainne; Taylor, Robert; Mead, Richard; Shaw, Pamela; Day, John; Dudgeon, Alexander; Kendall, Catherine; Healthcare ScientistsRaman spectroscopy shows promise as a biomarker for complex nerve and muscle (neuromuscular) diseases. To maximise its potential, several challenges remain. These include the sensitivity to different instrument configurations, translation across preclinical/human tissues and the development of multivariate analytics that can derive interpretable spectral outputs for disease identification. Nonnegative matrix factorisation (NMF) can extract features from high-dimensional data sets and the nonnegative constraint results in physically realistic outputs. In this study, we have undertaken NMF on Raman spectra of muscle obtained from different clinical and preclinical settings. First, we obtained and combined Raman spectra from human patients with mitochondrial disease and healthy volunteers, using both a commercial microscope and in-house fibre optic probe. NMF was applied across all data, and spectral patterns common to both equipment configurations were identified. Linear discriminant models utilising these patterns were able to accurately classify disease states (accuracy 70.2-84.5%). Next, we applied NMF to spectra obtained from the mdx mouse model of a Duchenne muscular dystrophy and patients with dystrophic muscle conditions. Spectral fingerprints common to mouse/human were obtained and able to accurately identify disease (accuracy 79.5-98.8%). We conclude that NMF can be used to analyse Raman data across different equipment configurations and the preclinical/clinical divide. Thus, the application of NMF decomposition methods could enhance the potential of Raman spectroscopy for the study of fatal neuromuscular diseases.Publication A review of kilovoltage radiotherapy treatment in the United Kingdom: quality control, radiation dosimetry, treatment equipment, and workload(British Institute of Radiology Oxford University Press, 2025-01-10) Palmer, Antony; Brimelow, Jason; Downes, Patrick; Munshi, Mayur; Nash, David; Rai, Bhupinder; White, Andrew; Brimelow, Jason; Additional Professional Scientific and TechnicalAbstract Objectives To survey kilovoltage (kV) radiotherapy in the United Kingdom, updating a 2016 study, focussing on radiotherapy physics, including equipment quality control (QC) and radiation dosimetry, with information on installed equipment and clinical activity. Methods All UK radiotherapy physics departments (n = 68) were invited to complete a comprehensive survey. An analysis of the installed equipment base, patient numbers, clinical activity, QC testing, and radiation dosimetry processes were undertaken. Results 91% of centres (n = 62) responded to the survey. kV radiotherapy was available in 70% of UK radiotherapy departments, with a wide variation in workload; 7-436 patients/centre annually. There has been an increase in centres using treatment calculation software rather than manual methods, up from 36% in 2016 to 50% currently. Only 50% of centres use an independent calculation check method. There was an increase in the use of the addendum to the UK dosimetry code of practice, enabling medium energy calibration in-air rather than at depth in phantom, citing “clinical relevance.” Appropriate levels of QC testing were being conducted at UK centres, with Institute of Physics and Engineering in Medicine (IPEM) Report 81 cited as a primary source of guidance. Good consensus for the frequency and tolerance values used for QC was seen across UK centres. Conclusions A comprehensive review of consensus practice for QC and dosimetry in kV radiotherapy across the United Kingdom is presented, with supporting information on equipment installation and clinical use. Advances in Knowledge Updated data are presented on kV radiotherapy treatment in the United Kingdom, with focus on physics aspects of QC and dosimetry.Publication Optimising Shifted Excitation Raman Difference Spectroscopy (SERDS) for application in highly fluorescent biological samples, using fibre optic probes(Royal Society of Chemistry, 2024-11-19) Sheridan, Hannah; Dudgeon, Alexander; Day, John; Kendall, Catherine; Hall, Charlie; Stone, Nick; Dudgeon, Alexander; Kendall, Catherine; Hall, Charlie; Stone, Nick; Healthcare Scientists; Medical and DentalFibre optic probe based Raman spectroscopy can deliver in vivo molecular compositional analysis of a range of diseases. However, some biological tissues exhibit high levels of fluorescence which limit the utility of the technique, particularly when the fluorescence induces CCD etaloning, which can be particulalry hard to remove in subsequent analysis. Furthermore, use of fibre probes can result in silica signals superimposed on the biological Raman signals. Shifted excitation Raman difference spectroscopy (SERDS) utilises a small seperation in excitation wavelengths to remove signals from fluorescence, room lights, optical components and etaloning contributions, while retaining chemical signals from the sample. In this study, we sought to measure the optimum SERDS spectra enabling reconstruction of a range a narrow and broad peaks found in biological samples. A original wavelength of 830 nm was utilised with 7 different shifts between 0.4 and 3.9 nm to determine which gave the best performance. This range roughly corresponds to the typical range of peak widths within biological Raman spectra at 830 nm excitation; 0.41 – 3.25 nm or 6 – 47 cm−1. An wavelength shift of 2.4 nm was identified as optimal. Finally, a fibre optic Raman probe was used to measure 2 human lymph nodes ex vivo to demonstrate the feasibility of the approach with real-world examples.Publication Conformational fingerprinting with Raman spectroscopy reveals protein structure as a translational biomarker of muscle pathology(Royal Society of Chemistry, 2024-03-27) Alix, James; Plesia, Maria; Dudgeon, Alexander; Kendall, Catherine; Hewamadduma, Channa; Hadjivassiliou, Marios; Gorman, Gráinne; Taylor, Robert; McDermott, Christopher; Shaw, Pamela; Mead, Richard; Day, John; Dudgeon, Alexander; Kendall, Catherine; Healthcare ScientistsNeuromuscular disorders are a group of conditions that can result in weakness of skeletal muscles. Examples include fatal diseases such as amyotrophic lateral sclerosis and conditions associated with high morbidity such as myopathies (muscle diseases). Many of these disorders are known to have abnormal protein folding and protein aggregates. Thus, easy to apply methods for the detection of such changes may prove useful diagnostic biomarkers. Raman spectroscopy has shown early promise in the detection of muscle pathology in neuromuscular disorders and is well suited to characterising the conformational profiles relating to protein secondary structure. In this work, we assess if Raman spectroscopy can detect differences in protein structure in muscle in the setting of neuromuscular disease. We utilise in vivo Raman spectroscopy measurements from preclinical models of amyotrophic lateral sclerosis and the myopathy Duchenne muscular dystrophy, together with ex vivo measurements of human muscle samples from individuals with and without myopathy. Using quantitative conformation profiling and matrix factorisation we demonstrate that quantitative 'conformational fingerprinting' can be used to identify changes in protein folding in muscle. Notably, myopathic conditions in both preclinical models and human samples manifested a significant reduction in α-helix structures, with concomitant increases in β-sheet and, to a lesser extent, nonregular configurations. Spectral patterns derived through non-negative matrix factorisation were able to identify myopathy with a high accuracy (79% in mouse, 78% in human tissue). This work demonstrates the potential of conformational fingerprinting as an interpretable biomarker for neuromuscular disorders.