Decomposition of in vivo spatially offset Raman spectroscopy data using multivariate analysis techniques

The decomposition of spatially offset Raman spectra for complex multilayer systems, such as biological tissues, requires advanced techniques such as multivariate analyses. Often, in such situations, the decomposition methods can reach their limits of accuracy well before the limits imposed by signal‐to‐noise ratios. Consequently, more effective reconstruction methods could yield more accurate results with the same data set. In this study we process spatially offset Raman spectroscopy (SORS) data with three different multivariate techniques (band‐target entropy minimization (BTEM), multivariate curve resolution and parallel factor analysis (PARAFAC)) and compare their performance when analysing a spectrally challenging plastic model system and an even more challenging problem, the analysis of human bone transcutaneously in vivo. For the in vivo measurements, PARAFAC's requirement of multidimensional orthogonal data is addressed by recording SORS spectra both at different spatial offsets and at different anatomical points, the latter providing added dimensionality through the variation of skin/soft tissue thickness. The BTEM and PARAFAC methods performed the best on the plastic system with the BTEM more faithfully reconstructing the major Raman bands and PARAFAC the smaller more heavily overlapped features. All three methods succeeded in reconstructing the bone spectrum from the transcutaneous data and gave good figures for the phosphate‐to‐carbonate ratio (within 2% of excised human tibia bone); the PARAFAC gave the most accurate figure for the mineral‐to‐collagen ratio (20% less than excised human tibia bone). Previous studies of excised bones have shown that certain bone diseases (such as osteoarthritis, osteoporosis and osteogenesis imperfecta) are accompanied by compositional abnormalities that can be detected with Raman spectroscopy, the utility of a technique which could reconstruct bone spectra accurately is manifest. The results have relevance on the use of SORS in general. © 2014 Crown copyright. Journal of Raman Spectroscopy published by John Wiley & Sons, Ltd.     

Raman Spectroscopy of Natural Bone and Synthetic Apatites

Abstract

Raman spectroscopy of natural bones and hydroxyapatites is described. In addition, how Raman spectroscopy has proved crucial in providing baseline data for the modification of synthetic apatite powders that are routinely used now as bone replacement materials is explained. It is important to understand the chemical structural properties of natural bone. Bone consists of two primary components: an inorganic or mineral phase, which is mainly a carbonated form of a nanoscale crystalline calcium phosphate, closely resembling hydroxyapatite, and an organic phase, which is composed largely of type I collagen fibers. Other constituents of bone tissue include water and organic molecules such as glycosaminoglycans, glycoproteins, lipids, and peptides. Ions such as sodium, magnesium, fluoride, and citrate are also present, as well as hydrogenophosphate. Hence, the mineral phase in bone may be characterized essentially as nonstoichiometric substituted apatite. Such a distinction is important in the development of synthetic calcium phosphates for application as skeletal implants. An understanding of bone function and its interfacial relationship to an implant clearly depends on the associated structure and composition. Therefore, it is essential to fully understand the chemical composition of bone, and Raman spectroscopy is an excellent technique for such an analysis.     

Towards the in vivo prediction of fragility fractures with Raman spectroscopy

Fragility fractures, those fractures which result from low level trauma, have a large and growing socio‐economic cost in countries with aging populations. Bone‐density‐based assessment techniques are vital for identifying populations that are at higher risk of fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone non‐invasively in vivo. Unlike X‐ray‐based techniques SORS can retrieve chemical information from both the mineral and protein phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture because both phases contribute to bone's mechanical properties. In this study we analyse excised bone with Raman spectroscopy and multivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excised work that on average, bone fragments from the necks of fractured femora are more mineralised (by 5–10%) than (cadaveric) non‐fractured controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy and diseased human cohorts), we identify methodological developments which will be used to improve the statistical significance of future experiments and may eventually lead to more sensitive prediction of fragility fractures. © 2015 The Authors. Journal of Raman Spectroscopy Published by John Wiley & Sons, Ltd.     

拉曼光谱在骨组织研究中的应用

拉曼光谱具有快速、无损、能同时提供无机和有机成分信息,包括矿物晶格和胶原蛋白二级结构信息等特点,在骨组织研究中有着重要应用。本文结合本课题组工作,对骨组织的光谱背景处理、拉曼光谱在骨的力学性能、骨科疾病、成像研究以及活体检测等方面的应用进行了综述。     

Chemical and mechanical properties of snake fangs

The composition of dental tissues and their interaction determines its mechanical properties. The mechanical properties and chemical composition of the teeth of extant reptiles are still poorly studied areas. As a preliminary study the fangs of four species of snakes and a human tooth were investigated through nanoindentation and Raman spectroscopy. The average elastic modulus values for the main body of the fangs ranged from 15.3 GPa to 24.6 GPa, and 19.1 GPa for the human dentine. Raman spectroscopy and principal component analysis (PCA) showed that snake fangs are similar in composition to human dentine, both of which comprised of hydroxyapatite and an organic matrix. The elastic modulus and hardness data were correlated to the Raman spectra using partial least squares regression (PLS). The spectral features which correlated with the elastic modulus would suggest that elastic modulus is dependent on the relative protein to mineral amounts in the tooth. The form of the phosphate and the relative levels of phosphate to organic components also appear to be governing factors for elastic modulus. The PLS of Raman spectra against the hardness gave very similar results. The small differences between snake fangs and human dentine appeared to be because of carbonate content, with higher levels of carbonate in the human tooth than the snake fangs. Snake fangs should be able to withstand large lateral forces. Human dentine aids in dissipating imposed loads. This similarity in the chemical composition of the snake fangs and human dentine supported the findings of the similarities in mechanical properties, which may be attributed to the similar functional demands of these biocomposites. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular structural analysis of noncarious cervical sclerotic dentin using Raman spectroscopy

Molecular structure of the sclerotic dentin in noncarious cervical lesions (NCCLs) including both the inorganic phase and organic phase was investigated using Raman spectroscopy. It was found that NCCL sclerotic dentin was hypermineralized with the mineral/matrix ratios 2–3 times higher than those of normal dentin, which was caused by both the increase of mineral content and decrease of organic matrix (collagen) content in the sclerotic dentin. For the inorganic phase, the phosphate band (PO₄³⁻, ν₁, symmetric stretching vibrational mode) in NCCL sclerotic dentin was shifted from 960 to 963 cm⁻¹, and the width of this band was decreased from 16.4 to 10.4 cm⁻¹, indicating that the degree of mineral crystallinity in NCCL sclerotic dentin was higher than that of normal dentin. In addition, the carbonate content in the mineral of NCCL sclerotic dentin was less than that of normal dentin. As compared to the inorganic phase, the changes within the organic phase were not dramatic. However, the changes in collagen cross‐link density along with other spectral changes were still detectable. There was a noteworthy reduction in the ratio of nonreducible to reducible cross‐links in the NCCL sclerotic dentin, indicating that cross‐link breaks occurred in the collagen matrix of the lesions. Copyright © 2009 John Wiley & Sons, Ltd.     

Raman spectroscopic study of synthetic reevesite and cobalt substituted reevesite (Ni,Co)6Fe2(OH)16(CO3)·4H2O

Raman spectroscopy, complemented with infrared spectroscopy of compounds equivalent to reevesite, formula (Ni,Co)₆Fe₂(OH)₁₆(CO₃)·4H₂O, with the ratio of Ni/Co ranging from 0 to 1, have been synthesised and characterised based on the molecular structure of the synthesised mineral. The combination of Raman spectroscopy with infrared spectroscopy enables an assessment of bands attributable to water stretching and brucite‐like surface hydroxyl units to be obtained. Raman spectroscopy shows a reduction in the symmetry of the carbonate anion, leading to the conclusion that the carbonate anion is bonded to the brucite‐like hydroxyl surface and to the water in the interlayer. Variation in the position of the carbonate anion stretching vibrations occurs and is dependent on the Ni/Co ratio. Water bending modes are identified in both the Raman and infrared spectra at positions greater than 1620 cm⁻¹, indicating that water is strongly hydrogen bonded to both the interlayer anions and the hydrotalcite surface. Copyright © 2009 John Wiley & Sons, Ltd.     

FT‐Raman spectroscopic study of thoracic aortic wall subjected to uniaxial stress

The combination of Fourier transform‐Raman spectroscopy and uniaxial tensile tests (in MTS Synergie 100 testing machine) was used to investigate microstructural changes in the secondary protein structure of the aortic wall under different levels of stress. The spectroscopic analysis clearly shows differing tension thresholds for material excised in two directions: circumferential and longitudinal. This is confirmed by the results of macroscopic mechanical analyses. The application of strain does not lead to any noticeable change in the bandwidths of the Raman bands. The stress‐controlled Raman band analysis shows that the modes at 938 cm⁻¹ assigned as C_α C of the α‐helix, 1660 cm⁻¹ amide I (the unordered structure of elastin) and 1668 cm⁻¹ amide I (the collagen triple helix) undergo wavenumber shifting, but the bands at 1004 cm⁻¹ assigned to the phenyl ring breathing mode and 2940 cm⁻¹ to the ν (CH₃) and ν (CH₂) modes are not affected during the elastic behaviour. A clear correlation between Raman band shifting and the level of mechanical stress has been established. Elastin alone participates in the transmission of low stresses in the circumferential direction, whereas both elastin and collagen take part in the transmission of physiological and higher stresses. Copyright © 2009 John Wiley & Sons, Ltd.     

Raman spectroscopy of dipyrrins: nonresonant,resonant and surface‐enhanced cross‐sections and enhancement factors

Detailed studies of the mechanism of surface‐enhanced (resonance) Raman spectroscopy (SE(R)RS), and its applications, place a number of demands on the properties of SERS scatterers. With large Raman cross‐sections, versatile synthetic chemistry and complete lack of fluorescence, free dipyrrins meet these demands but the Raman and SE(R)RS spectroscopy of free dipyrrins is largely unknown. The first study of the Raman spectroscopy of free dipyrrins is therefore presented in this work. The nonresonant Raman, resonant Raman and surface‐enhanced Raman spectra of a typical meso aryl‐substituted‐dipyrrin are reported. Absolute differential cross‐sections are obtained for excitation wavelengths in the near infrared and visible region, in solution phase and for dipyrrin adsorbed on the surface of silver nanoparticles. Raman enhancement factors for SERRS and resonance Raman are calculated from the observed differential cross‐sections. The magnitudes of the resonantly enhanced cross‐sections are similar to those recently reported for strong SERS dyes such as Rhodamine 6G and Crystal Violet. Free dipyrrins offer the advantages of existing SERS dyes but without the drawback of strong fluorescence. Free dipyrrins should therefore find applications in all areas of Raman spectroscopy including fundamental studies of the mechanisms of SERS and bioanalytical and environmental applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Raman and surface‐enhanced Raman scattering in the study of human rotator cuff tissues after shock wave treatment

Important improvements of diseases of the rotator cuff supraspinatus tendons are seen after shock wave (SW) treatment. Neo‐angiogenesis stimulation and hypercellularization result from short periods of treatment. The present work is an attempt to provide a first approach to these bioprocesses, most likely associated with structural aspects resulting from biochemical changes brought about by the SW. Immunohistochemical data indicate that collagen areas in the tissues are influenced the most by the SW. Presence of additional collagens I and III by the SW treatment is inferred from an observed increase of the tissue's tinctorial properties. The tools selected for our studies are Raman spectroscopy and the ultrasensitive surface‐enhanced Raman scattering (SERS). Here we extract information from 1016 SERS spectra of 52 biopsies of human tendon tissues on Ag nanoparticles before and after the SW treatment. The spectral information is analyzed on the basis of Raman and SERS data of collagen types I and III and their most abundant amino acid components. SERS spectra of tissues reveal the presence of characteristic modes related mainly to amino acids. It has been found that the main differences between both tissue samples could be correlated with the structural conformational aspects of collagen. Copyright © 2011 John Wiley & Sons, Ltd.     

Applications of Raman Spectroscopy in Dentistry: Analysis of Tooth Structure

Abstract: Tooth enamel is the most mineralized tissue in the human body, and in this article the use of Raman spectroscopy for the analysis of tooth structure, a comparison with synthetic apatites, and use in dentistry are described. Spectral peaks that are related to dental hard and soft tissues are discussed, which provide crucial data in understanding the chemical structural properties of dentin and enamel. The Raman spectrum of dentin confirms the presence of crystalline phosphate-based minerals in dentin. Both dentin and enamel consist of two primary components: an inorganic or mineral phase that closely resembles hydroxyapatite and the Raman spectrum of dentin that confirms the presence of crystalline phosphate-based minerals in dentin. Hence, the mineral phase in dentin and enamel may be characterized essentially as nonstoichiometric substituted apatite. The presence of carbonate (A and B type) incorporated in the hydroxyapatite lattice is also confirmed by the presence of spectral bands. The organic phase, which is mainly composed of type I collagen, is confirmed by the spectral bands of amide I and amide II bands, tryptophan, and phenylalanine. Furthermore, these spectral bands associated with organic and inorganic parts of the enamel and dentin are useful in predicting early formation of carries formation.     

A Raman spectroscopic study of teeth affected with molar–incisor hypomineralisation

Raman spectroscopy was used to chemically map lesions associated with molar–incisor hypomineralisation in human teeth. Three teeth with hypomineralised lesions of differing severity, described as white, yellow or brown, were mapped using integral ratios of major component bands (hydroxyapatite, amide I and b‐type carbonate) and principal component analysis scores values. These lesions were found to contain depleted levels of mineral (hydroxyapatite) compared with those of healthy enamel. Principal component analysis also highlighted changes in the phosphate structure and variations in various organic constituents. These variations were consistent with increased disorder in the mineral component of the hypomineralised tooth lesions. Scanning electron microscopy–energy dispersive X‐ray spectroscopy supported the findings based on Raman spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Mineralogical changes of bioapatite in femoral bones of mice during pregnancy

Mineralogical changes and solubility of bioapatite in mice cortical bones (2- and 5 months old) were investigated by Raman spectroscopy and inductively coupled plasma spectrometry in this study. The Raman analyses show that the full width at half maximum values of the 960?cm^?1 peak exhibit a slight increase during mouse pregnancy, which suggests slightly decreasing crystallinity. Additionally, aging significantly increases the solubility of bone bioapatite. The 1070?cm^?1 peak indicates the enriched carbonate incorporation during aging from 2 to 5 months. The elevated solubility of bioapatite and low bone density are consistent with the low crystallinity of bioapatite.     

Crocoite PbCrO4 and mimetite Pb5(AsO4)3Cl: rare minerals in highly degraded mediaeval murals in Northern Bohemia

Mural paintings of exceptional quality, which can be discerned in spite of their extensive mechanical damage and colour fading, have been uncovered in the church of St. Gallus in Kuřívody, Northern Bohemia, dated to the second half of the 13th century. Materials research with particular use of portable X‐ray fluorescence, Raman micro‐spectroscopy and powder X‐ray micro‐diffraction revealed the presence of rare pigments. In Kuřívody, it is only a second identification of intentionally used yellow mineral crocoite (PbCrO₄) in European art. Its identification is facilitated by providing a very good Raman scattering, even when present in small amounts in fragmentarily preserved colour layers. Light yellow mimetite (Pb₅(AsO₄)₃Cl) was never before mentioned as intentionally used pigment in Europe. Its finding in Kuřívody, however, corresponds more likely with undesirable physical–chemical conditions causing its formation by alteration of orpiment (As₂S₃) and minium (Pb₃O₄). Obtained results highlight the importance of Raman spectroscopy for direct identification of mineral pigments in low concentrations, which may be crucial for interpreting cultural heritage objects in historical context. By materials, the almost forgotten paintings in Kuřívody can be seen as outstanding and rare example of ancient artistic tradition that has spread to Europe from Mediterranean in early Middle Ages. After all, mineral crocoite was already used by ancient Egyptians to paint sarcophagi and degraded orpiment decorates the walls of the Nefertari's tomb in Thebes. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of Biot's theory to ultrasonic characterization of human cancellous bones: determination of structural, material, and mechanical properties

This paper is devoted to the experimental determination of distinctive macroscopic structural (porosity, tortuosity, and permeability) and mechanical (Biot-Willis elastic constants) properties of human trabecular bones. Then, the obtained data may serve as input parameters for modeling wave propagation in cancellous bones using Biot's theory. The goal of the study was to obtain experimentally those characteristics for statistically representative group of human bones (35 specimens) obtained from a single skeletal site (proximal femur). The structural parameters were determined using techniques devoted to the characterization of porous materials: electrical spectroscopy, water permeametry, and microcomputer tomography. The macroscopic mechanical properties, Biot-Willis elastic constants, were derived based on the theoretical consideration of Biot's theory, micromechanical statistical models, and experimental results of ultrasonic studies for unsaturated cancellous bones. Our results concerning structural parameters are consistent with the data presented by the other authors, while macroscopic mechanical properties measured within our studies are situated between the other published data. The discrepancies are mainly attributed to different mechanical properties of the skeleton frame, due to strong structural anisotropy varying from site to site. The results enlighten the difficulty to use Biot's theory for modeling wave propagation in cancellous bone, implying necessity of individual evaluation of input parameters.     

Multiphonon Raman spectroscopy properties and Raman mapping of 2D van der Waals solids: graphene and beyond

Strong in‐plane bonding (covalent) and weak van der Waals (vdW) interplanar interactions characterize a number of layered solids, as epitomized by graphite. The advent of graphene (Gr), individual atomic two‐dimensional (2D) layers, isolated from mineral graphite via micromechanical exfoliation enabled the ability to pick, place and stack of arbitrary compositions. Moreover, this discovery implicated an access to other 2D vdW solids beyond graphene and artificially stacking atomic layers forming heterostructures/superlattices. Raman spectroscopy (RS) is a fast reliable non‐destructive analytical tool and an integral part for lattice dynamical structural characterization of crystalline solids at nanoscale, revealing not only the collective atomic/molecular motions but also localized vibrations/modes and specifically used to determine the number of graphene layers and of other 2D vdW solids. We present Raman spectroscopy in first‐, second‐ and higher‐order vibrational modes involving 3 and 4 phonons (overtones and combination) and mapping of graphene (mono‐, bi‐, tri‐ and few‐) layers, semiconducting transition metal dichalcogenides (TMDs) [molybdenum disulfide (MoS₂) and tungsten disulfide (WS₂)] and wide bandgap hexagonal boron nitride (h‐BN) dispersed monolayers, revealing various molecular vibrations and structural quality/disorder. First‐ and higher‐order phonon modes are observed and analyzed in terms of Raman intensity (spatial inhomogeneity or thickness variation), band position (intrinsic mechanical strain) and intensity ratio (structural disorder) as a function of graphene layer (n). An empirical relation for G band position with n is corroborated. All of the higher order modes are observed to upshift almost linearly with n, betraying the underlying interlayer vdW interactions. These findings exemplify the evolution of structural parameters in layered materials in changing from 3‐ to 2‐ or low‐dimensional regime. The results are presented in view of applications of graphene by itself and in combination that help better understanding of physical and electronic properties for nano‐/optoelectronics. Copyright © 2015 John Wiley & Sons, Ltd.     

基于拉曼光谱的复杂硫化矿快速分类方法研究

热液硫化物型脉状矿作为一类复杂硫化矿,其区域特征、成矿规律及矿物成份已有初步研究。由于成矿时期的不同,矿石中有用矿物的特征存在较大差异,导致不同矿物的性质变化较大。在选矿过程中,矿物性质的差异一定程度增加了选矿难度,减少了有用矿物回收率。因此,迫切需要一种快速、简单的对复杂硫化矿进行分类的方法,进而提高选矿指标。激光拉曼光谱技术作为一种能够分析物质结构信息的手段,已被应用于矿物的成份鉴定和结构分析。通过对大量矿物样本的激光拉曼光谱的研究,结合矿物性质深入揭示其光谱差异的原因,提出了一种基于拉曼光谱的复杂硫化矿矿源分类方法。实验结果表明：由于此类复杂硫化矿成矿时期的差异,从而造成矿物结构和性质存在较大差异。荧光主要由原矿中的脉石矿物产生,猝灭矿物中瞒石的荧光背景后可知201.62, 242.54, 288.38和309.77 cm-1处拉曼峰可以作为此类硫化矿的拉曼指纹谱。为此,基于此类硫化矿的荧光强度和代表谱峰强度与荧光背景比值可以将矿源分为三类,并利用工业试验结果进一步验证分类方法的准确性。本研究深入分析了此类复杂硫化矿的激光拉曼光谱与其矿物性质与类别之间的密切关系,提出了矿源快速分类方法,矿样无需经过复杂的化学前处理过程,对提高选矿作业效率具有重要应用价值。     

Investigations on the geometrical isomers of astaxanthin: Raman spectroscopy of conjugated polyene chain with electronic and mechanical confinement

Recent research interests in geometrical isomers of astaxanthin (AST) are motivated by their metabolic activities in aquatic animals and human. It has been established that cis‐isomers of AST are selectively absorbed in human plasma during the metabolic process; however, exact absorption mechanism is still unclear. Hence, a detailed investigation of the structural and optical properties of geometrical isomers of AST is required. Among the techniques available for the study of AST and other carotenoids, Raman spectroscopy has been much acclaimed. Raman spectra have been shown to be influenced by the electronic and mechanical confinement effects arising from the conjugated polyene chain of carotenoids. In this work, we present Raman studies of geometrical isomers of AST, along with their optical absorption characteristics. Geometrical isomers of AST were prepared by heating all trans‐AST in solution form, and the isomers were separated using high performance liquid chromatography. Optical absorption spectra of cis‐isomers of AST showed hypsochromic shifts in the main absorption band and formation of new bands at lower wavelengths. A detailed Raman spectral analysis performed on the cis‐isomers of AST showed new modes which have not been observed and accounted for so far. In addition, we demonstrate that the electronic and mechanical confinement effects in the polyene chain of AST play an important role in the Raman spectra of geometrical isomers of AST. It is anticipated that this work will demonstrate that Raman spectroscopy is an important diagnostic tool in distinguishing and identifying the geometrical isomers of AST. Copyright © 2014 John Wiley & Sons, Ltd.     

Raman analysis of proteoglycans simultaneously in bone and cartilage

Bone and cartilage are connective tissues with distinct organic matrix (collagen and non‐collagenous proteins) composition facilitating their biological function. Proteoglycans (PGs), a member of the non‐collagenous proteins fulfill functions that are determined by both their core protein and their glycosaminoglycan chains. The purpose of the present study was to identify Raman bands that are representative of PG concentration and may be used in both bone and cartilage tissues. To achieve this goal, we analyzed a series of reference PGs and collagens, as well as turkey leg tendon to verify the laser polarization independency of the identified bands. Additionally, the applicability of these bands in both cartilage and bone tissue simultaneously was tested in a healthy femoral head by Raman imaging and hierarchical cluster analysis to describe the distribution of PGs at the micron level from articular cartilage to subchondral bone. The results of the study show that the Raman band ~1375 cm⁻¹ can be used as a PGs marker band in both cartilage and bone. Moreover, articular cartilage has a lower content of organic matrix (mostly type II collagen), while the middle and deep transitional zone haves a higher concentration of PGs. The calcified cartilage is characterized by a lower content of PGs and total organic matrix (estimated from the integrated area of the amide III band). Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo study on the protection of indole‐3‐carbinol (I3C) against the mouse acute alcoholic liver injury by micro‐Raman spectroscopy

Micro‐Raman spectroscopy (MRS) was utilized for the first time to evaluate the effect of indole‐3‐carbinol (I3C) on acute alcoholic liver injury in vivo. In situ Raman analysis of tissue sections provided distinct spectra that can be used to distinguish alcoholic liver injury as well as ethanol‐induced liver fibrosis from the normal state. Sixteen mice with liver diseases including acute liver injury and chronic liver fibrosis, and eight mice with normal liver tissues, and eight remedial mice were studied employing the Raman spectroscopic technique in conjunction with biomedical assays. The biochemical changes in mouse liver tissue when liver injury/fibrosis occurs such as the loss of reduced glutathione (GSH), and the increase of collagen (α‐helix protein) were observed by MRS. The intensity ratio of two Raman peaks (I₁₄₅₀/I₆₆₆) and in combination with statistical analysis of the entire Raman spectrum was found capable of classifying liver tissues with different pathological features. Raman spectroscopy therefore is an important candidate for a nondestructive in vivo screening of the effect of drug treatment on liver disease, which potentially decreases the time‐consuming clinical trials. Copyright © 2008 John Wiley & Sons, Ltd.