Raman Spectroscopic Studies on Screening of Myopathies Rekha Gautam, † Sandeep Vanga, † Aditi Madan, ‡ Narayanappa Gayathri, § Upendra Nongthomba,* ,‡ and Siva Umapathy* ,†,∥ † Department of Inorganic and Physical Chemistry, ‡ Department of Molecular Reproduction, Development and Genetics, and ∥ Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore, 560012, India § National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore, 560029, India * S Supporting Information ABSTRACT: Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld 1 (up 1 ), heldup 2 (hdp 2 ), myosin heavy chain 7 (Mhc 7 ), actin88F KM88 (Act88F KM88 ), upheld 101 (up 101 ), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and β-sheet and/or random coil protein content along with a decrease in α-helix protein. Interestingly, the 12th day samples of up 1 and Act88F KM88 showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up 1 and Act88F KM88 (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up 101 and hdp 2 (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up 1 ). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy. M yopathies or muscle disorders may arise due to many factors, such as myofibrillar and internal cytoskeletal protein gene mutations, infection, nutrient deficiency, and so forth. 1 These disorders can be broadly divided into two groups: (i) the neuromuscular diseases, which include Dystrophies, Spinalatrophies, Parkinson’s, and so forth; and (ii) the musculoskeletal diseases, which include disorders such as cardiomyopathies, nemaline-myopathies, and so forth. 1−4 Globally, such muscle-related disorders are known to be one of the leading causes of fatality. 5 Usually, diagnosis of myopathies involves numerous tests, including but not limited to muscle strength analyses using electromyography, ultrasound, and magnetic resonance imaging (MRI), which detect electrical activity of the muscle, muscle inflammation, abnormal muscle, and so forth. 4−7 However, none of these techniques are able to provide chemical (molecule) specific information on the pathology to be studied. The traditional pathologist’s inter- pretations of muscle biopsies generally rely only on the visual morphological changes. Myopathies are a clinically and genetically heterogeneous group of diseases with a wide spectrum of phenotypes. Thus, they sometimes remain undiagnosed until they progress to a point at which the disorder can obviously be identified but has become difficult to treat effectively. 4,8 Although there are numerous studies on the basis of genetics, a complete understanding of the pathophysiology from a molecular point of view is still lacking, which hampers the development of a suitable therapy. Furthermore, since there is no effective and complete cure for any of the myopathies or dystrophies, a sensitive and early diagnosis of these disorders is a necessity. 4 Therefore, a fast, reliable, sensitive, and specific screening technology for disease detection is needed. Raman spectroscopy, a label free, noninvasive, and multi- plexing modality, is an emerging analytical technique in biomedical research. 9−12 As an analytical tool, Raman spectro- scopic methods provide molecular structure and conformation- dependent spectral markers of the chemical components in a heterogeneous sample. This method is based on the principle of inelastic scattering of monochromatic (laser) light. Upon interaction with molecules, the laser light generates scattered radiation of different wavelengths that together provides a fingerprint spectrum of the molecular structure. The Raman Received: September 23, 2014 Accepted: January 12, 2015 Published: January 12, 2015 Article pubs.acs.org/ac © 2015 American Chemical Society 2187 DOI: 10.1021/ac503647x Anal. Chem. 2015, 87, 2187−2194