Combining electromyography and Raman spectroscopy: optical EMG.

INTRODUCTION/AIMS: Electromyography (EMG) remains a key component of the diagnostic work-up for suspected neuromuscular disease, but it does not provide insight into the molecular composition of muscle which can provide diagnostic information. Raman spectroscopy is an emerging neuromuscular biomarker capable of generating highly specific, molecular fingerprints of tissue. Here, we present "optical EMG," a combination of EMG and Raman spectroscopy, achieved using a single needle. METHODS: An optical EMG needle was created to collect electrophysiological and Raman spectroscopic data during a single insertion. We tested functionality with in vivo recordings in the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), using both transgenic (n = 10) and non-transgenic (NTg, n = 7) mice. Under anesthesia, compound muscle action potentials (CMAPs), spontaneous EMG activity and Raman spectra were recorded from both gastrocnemius muscles with the optical EMG needle. Standard concentric EMG needle recordings were also undertaken. Electrophysiological data were analyzed with standard univariate statistics, Raman data with both univariate and multivariate analyses. RESULTS: A significant difference in CMAP amplitude was observed between SOD1G93A and NTg mice with optical EMG and standard concentric needles (p = .015 and p = .011, respectively). Spontaneous EMG activity (positive sharp waves) was detected in transgenic SOD1G93A mice only. Raman spectra demonstrated peaks associated with key muscle components. Significant differences in molecular composition between SOD1G93A and NTg muscle were identified through the Raman spectra. DISCUSSION: Optical EMG can provide standard electrophysiological data and molecular Raman data during a single needle insertion and represents a potential biomarker for neuromuscular disease.

Label-free fibre optic Raman spectroscopy with bounded simplex-structured matrix factorization for the serial study of serum in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease in urgent need of disease biomarkers for the assessment of promising therapeutic candidates in clinical trials. Raman spectroscopy is an attractive technique for identifying disease related molecular changes due to its simplicity. Here, we describe a fibre optic fluid cell for undertaking spontaneous Raman spectroscopy studies of human biofluids that is suitable for use away from a standard laboratory setting. Using this system, we examined serum obtained from patients with ALS at their first presentation to our centre (n = 66) and 4 months later (n = 27). We analysed Raman spectra using bounded simplex-structured matrix factorization (BSSMF), a generalisation of non-negative matrix factorisation which uses the distribution of the original data to limit the factorisation modes (spectral patterns). Biomarkers associated with ALS disease such as measures of symptom severity, respiratory function and inflammatory/immune pathways (C3/C-reactive protein) correlated with baseline Raman modes. Between visit spectral changes were highly significant (p = 0.0002) and were related to protein structure. Comparison of Raman data with established ALS biomarkers as a trial outcome measure demonstrated a reduction in required sample size with BSSMF Raman. Our portable, simple to use fibre optic system allied to BSSMF shows promise in the quantification of disease-related changes in ALS over short timescales.

Rapid identification of human muscle disease with fibre optic Raman spectroscopy.

The 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.

Developing fibre optic Raman probes for applications in clinical spectroscopy.

Raman spectroscopy has been shown by various groups over the last two decades to have significant capability in discriminating disease states in bodily fluids, cells and tissues. Recent development in instrumentation, optics and manufacturing approaches has facilitated the design and demonstration of various novel in vivo probes, which have applicability for myriad of applications. This review focusses on key considerations and recommendations for application specific clinical Raman probe design and construction. Raman probes can be utilised as clinical tools able to provide rapid, non-invasive, real-time molecular analysis of disease specific changes in tissues. Clearly the target tissue location, the significance of spectral changes with disease and the possible access routes to the region of interest will vary for each clinical application considered. This review provides insight into design and construction considerations, including suitable probe designs and manufacturing materials compatible with Raman spectroscopy.

Characterisation of a fibre optic Raman probe within a hypodermic needle.

We demonstrate the first use of a multifibre Raman probe that fits inside the bore of a hypodermic needle. A Raman probe containing multiple collection fibres provides improved signal collection efficiency in biological samples compared with a previous two-fibre design. Furthermore, probe performance (signal-to-noise ratios) compared favourably with the performance achieved in previous Raman microscope experiments able to distinguish between benign lymph nodes, primary malignancies in lymph nodes and secondary malignancies in lymph nodes. The experimental measurements presented here give an indication of the sampling volume of the Raman needle probe in lymphoid tissues. Liquid tissue phantoms were used that contained scattering medium encompassing a range of scattering properties similar to those of a variety of tissue types, including lymph node tissues. To validate the appropriateness of the phantoms, the sampling depth of the probe was also measured in excised lymph node tissue. More than 50 % of Raman photons collected were found to originate from between the tip of the needle and a depth of 500 µm into the tissue. The needle probe presented here achieves spectral quality comparable to that in numerous studies previously demonstrating Raman disease discrimination. It is expected that this approach could achieve targeted subcutaneous tissue measurements and be viable for use for the in vivo Raman diagnostics of solid organs located within a few centimetres below the skin's surface. Graphical Abstract Schematic of multi-fibre Raman needle probe with disposible tips and proximal optical filtration.

In Vivo Fiber Optic Raman Spectroscopy of Muscle in Preclinical Models of Amyotrophic Lateral Sclerosis and Duchenne Muscular Dystrophy.

Neuromuscular 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.

A subcutaneous Raman needle probe.

Raman spectroscopy is a powerful tool for studying the biochemical composition of tissues and cells in the human body. We describe the initial results of a feasibility study to design and build a miniature, fiber optic probe incorporated into a standard hypodermic needle. This probe is intended for use in optical biopsies of solid tissues to provide valuable information of disease type, such as in the lymphatic system, breast, or prostate, or of such tissue types as muscle, fat, or spinal, when identifying a critical injection site. The optical design and fabrication of this probe is described, and example spectra of various ex vivo samples are shown.

Assessment of a custom-built Raman spectroscopic probe for diagnosis of early oesophageal neoplasia.

We evaluate the potential of a custom-built fiber-optic Raman probe, suitable for in vivo use, to differentiate between benign, metaplastic (Barrett's oesophagus), and neoplastic (dysplastic and malignant) oesophageal tissue ex vivo on short timescales. We measured 337 Raman spectra (λ(ex)=830 nm; P(ex)=60 mW; t=1 s) using a confocal probe from fresh (298) and snap-frozen (39) oesophageal tissue collected during surgery or endoscopy from 28 patients. Spectra were correlated with histopathology and used to construct a multivariate classification model which was tested using leave one tissue site out cross-validation in order to evaluate the diagnostic accuracy of the probe system. The Raman probe system was able to differentiate, when tested with leave one site out cross-validation, between normal squamous oesophagus, Barrett's oesophagus and neoplasia with sensitivities of (838% to 6%) and specificities of (89% to 99%). Analysis of a two group model to differentiate Barrett's oesophagus and neoplasia demonstrated a sensitivity of 88% and a specificity of 87% for classification of neoplastic disease. This fiber-optic Raman system can provide rapid, objective, and accurate diagnosis of oesophageal pathology ex vivo. The confocal design of this probe enables superficial mucosal abnormalities (metaplasia and dysplasia) to be classified in clinically applicable timescales paving the way for an in vivo trial.