Comparison of molecular images as defined by Raman spectra between normal mucosa and squamous cell carcinoma in the oral cavity

Raman spectroscopy has been effectively applied to clinically differentiate normal and cancerous mucosal tissues. Micro‐Raman spectroscopy provides a tool to better understand the molecular basis for the Raman clinical signal. The objective of the current study was to utilize micro‐Raman spectroscopy to define the molecular/spectral differences between normal and abnormal squamous cell carcinoma (SCC) in oral mucosa (in vitro). Understanding this may help in identifying unique spectra or may be useful for in vivo application of this technology. Micro‐Raman (confocal) spectroscopy was used to obtain molecular images of normal and SCC cells of human oral mucosa. Four fresh flashed‐frozen tumor and four matched normal tongue specimens were studied. The spectra covered a wavenumber range from 300 to 4000 cm⁻¹ with a spectral resolution of 8 cm⁻¹ and a spatial resolution of 1.0 µm. The cells were located within thin sections of tongue mucosa biopsies. The excitation wavelength of 515 nm was used. We were able to obtain Raman images with rich information about the spectroscopic and structural features within the cytoplasm, cell membrane, and cell nuclei. Significant spectral differences were observed between the Raman images of normal and malignant squamous cells. The heterogeneity of tumor cells within the abnormal tissue was also demonstrated. Spectral differences demonstrated between both tissue types have provided important information regarding the origins of specific signals within the cells of each tissue type. In our search for specific spectral biomarkers, we believe that a cell surface protein, greatly upregulated in SCC cells, was discovered at 1583 cm⁻¹. Copyright © 2010 John Wiley & Sons, Ltd.     

Evaluation of Raman spectroscopy for prediction of antitumor response to silibinin and its nanoparticulates in DMBA‐induced oral carcinogenesis

Raman spectroscopy is a vibrational spectroscopic technique that can be used to monitor the therapeutic efficacy of anticancer drugs during carcinogenesis in a non‐invasive and label‐free manner. The present study aims to investigate the biochemical changes exerted upon free silibinin (SIL) and its nanoparticulate (SILNPs) treatment against 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced oral carcinogenesis in the fingerprint region of 1800–500 cm⁻¹ using HE‐785 Raman spectrometer. Raman spectra differed significantly between the control and tumor tissues, with tumor tissues characterized by increased intensities of vibrational bands such as nucleic acids, phenylalanine and tryptophan and a lower percentage of lipids when compared to the control tissues. Further, oral administration of free SIL and SILNPs significantly increased lipids and decreased the levels of tryptophan, phenylalanine and nucleic acid contents. Overall, the treatment of nanoparticulate SIL was found to be a more potent antitumor effect than free SIL in preventing the formation of tumor and also brought back the several Raman bands to a normal range in the buccal mucosa of hamsters during DMBA‐induced oral carcinogenesis. In addition, the detailed secondary structure of proteins in the control and experimental groups is also presented. Furthermore, the diagnostic algorithms based on principal component linear discriminant analysis (PC‐LDA) achieved an overall sensitivity of 94–100% and specificity of 76–100%. These results further demonstrate that Raman spectroscopy associated with PC‐LDA diagnostic algorithms could be a valuable tool for rapid and sensitive detection of specific biomolecular changes at the molecular level in response to anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.     

Method development: Raman spectroscopy‐based histopathology of oral mucosa

An earlier and more accurate detection of (small) cancerous and precancerous lesions in the oral cavity is essential to improve the prognosis of oral squamous cell carcinomas. Raman spectroscopy is being pursued as a potential method to realize this improvement, since the technique provides objective information on a biochemical level and can be used for real‐time guidance of the diagnostic procedure. Since oral mucosal tissue is inhomogeneous and comprises different layers and histological structures, a good understanding of the signal contributions of the individual layers and structures is required for an accurate interpretation of in vivo tissue spectra measurement volumes. The aim of this study was to create a standardized method to collect and analyse the spectral contributions of individual histopathological structures in oral mucosa. The method is based on Raman microspectroscopic mapping of unstained frozen tissue sections and subsequent histopathological annotation of the features in the resulting Raman images. The obtained annotated reference spectra were used as input in an unsupervised hierarchical cluster analysis in order to determine the spectral characteristics and variance within one histo(patho)logical structure. The described method resulted in an annotated database of Raman spectral characteristics of individual histopathological structures encountered in oral tissue. This database can be used as input for the development of classification and quantification algorithms, in order to achieve a high specificity and sensitivity for clinical diagnostic instruments. Additionally, this database can be used to optimize the exact location and measurement volume of in vivo measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

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 monitoring of the cellular activities of a tissue‐engineered ex vivo produced oral mucosal equivalent

To ensure quality control and assurance in tissue engineering, noninvasive, real‐time and aseptic evaluation of cell‐based devices is required before product release. In this study, Raman spectroscopy was applied to monitor the cellular activities of an oral mucosa equivalent (EVPOME) produced ex vivo from cultured autogenous oral keratinocytes and acellular dermis—AlloDerm. Raman spectra showed a positive correlation of the peak area ratio of amide I (1655 cm⁻¹)/phenylalanine (1004 cm⁻¹) with a negative linear regression (R² > 0.95) according to the number of cultured days, especially on the 14thand 21st day. This work demonstrates the successful application of Raman spectroscopy for quantitatively monitoring and evaluating the maturity of EVPOME. Copyright © 2010 John Wiley & Sons, Ltd.     

Raman spectroscopy in experimental oral carcinogenesis: investigation of abnormal changes in control tissues

Oral cancer is a major cause of mortality in South Asian men owing to rampant tobacco abuse. Cancers are also reported in non‐tobacco habitués, especially women, attributed to chronic irritations from irregular/sharp teeth, improper fillings, and poorly fit dentures. Conventional screening approaches are shown to be effective for high‐risk groups (tobacco/alcohol habitués). Raman spectroscopy (RS) is being extensively explored as an alternate/adjunct tool for diagnosis and management of oral cancers. In a previous Raman study on sequential oral carcinogenesis using hamster buccal pouch model, misclassifications between spectra from control and carcinogen [7,12‐dimethylbenz(a)anthracene (DMBA)]‐treated tissues were observed. Histopathology of some control tissues suggested pathological changes, attributable to repeated forceps‐induced irritations/trauma during animal handling. To explore these changes, in the present study, we recorded spectra from three different types of controls – vehicle control (n = 45), vehicle contralateral (n = 45), and DMBA contralateral (n = 70) – exposed to varying degree of forceps handling, along with DMBA‐treated pouches (n = 70) using a 14‐week carcinogenesis protocol. Spectra certified on the basis of histopathology and abnormal cell proliferation (cyclin D1 expression) were used to build models that were evaluated by independent test spectra from an exclusive set of DMBA‐treated and control animals. Many DMBA‐contralateral, vehicle‐control, and vehicle‐contralateral spectra were identified as higher pathologies, which subsequently corroborated with histopathology/cyclin D1 expression. Repeated forceps‐mediated injuries/irritations, during painting and animal handling, may elicit inflammatory responses, leading to neoplasm. The findings of the study suggest that RS could identify micro‐changes. Further, RS‐based in vivo imaging can serve as a promising label‐free tool for screening even in the non‐habitué population where conventional screening is shown to be not effective. Copyright © 2016 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.     

Photon migration of Raman signal in bone as measured with spatially offset Raman spectroscopy

Spatially offset Raman spectroscopy (SORS) is currently being developed as an in vivo tool for bone disease detection, but to date, information about the interrogated volume as influenced by the light propagation and scattering characteristics of the bone matrix is still limited. This paper seeks to develop our general understanding of the sampling depths of SORS in bone specimens as a function of the applied spatial offset. Equine metacarpal bone was selected as a suitable specimen of compact cortical bone large enough to allow several thin slices (600 µm) to be cut from the dorsal surface. Photon migration at 830‐nm excitation was studied with five bone slices and a 380‐µm‐thin polytetrafluoroethylene (PTFE) slice placed consecutively between the layers. To optimize Raman signal recovery of the PTFE with increasing depth within the bone stack required a corresponding increase in spatial offset. For example, to sample effectively at 2.2‐mm depth within the bone required an optimal SORS offset of 7 mm. However, with a 7‐mm offset, the maximum accessible penetration depth from which the PTFE signal could be still recovered was 3.7 mm. These results provide essential basic information for developing SORS technology for medical diagnostics in general and optimizing sampling through bone tissue, permitting a better understanding of the relationship between the offset and depth of bone assessed, in particular. Potential applications include the detection of chemically specific markers for changes in bone matrix chemistry localized within the tissue and not present in healthy bone. © 2015 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons, Ltd.     

Do formalin fixation and freeze‐thaw affect near‐infrared Raman spectroscopy of cartilaginous tissue? A preliminary ex vivo analysis of native human articular cartilage

Near‐infrared (NIR) Raman microprobe spectroscopy has been applied to the non‐invasive characterization of the biochemical structure of extracellular matrix in articular cartilage, a step forward along the path of in vivo diagnostic application of chondropathy. In most studies handling ex vivo cartilage specimens, formalin fixation or freeze‐thaw treatments have been applied in order to stabilize tissue and cell constituents prior to spectroscopic measurements. However, these pre‐processing manipulations might significantly affect certain target bands of the cartilage spectra, thus introducing biases in the characterizations, and potentially leading to data misinterpretation. In this study, we evaluated how formalin fixing and freeze‐thaw processes affect Raman spectra from human femur cartilage. Healthy cartilage specimens were fixed/stored either in a 10% neutral buffered formalin solution or in a deep freezer set at −80 °C. The results of this study show that formalin fixation significantly affects the NIR Raman spectra of cartilage specimens due to concurrent formalin absorption and water dehydration within both collagen and glycosaminoglycan macromolecules. Water dehydration was also confirmed in the amide I structure in the frozen‐thawed specimen, but to a much lesser extent. Furthermore, soaking the tissues in phosphate‐buffered saline solution minimized the storage‐induced Raman artifacts, but its immersion had limited effectiveness in formalin‐fixed specimens, predominantly due to an overlap of signals from the formalin liquid (i.e. emitting at 1046 and 1492 cm⁻¹). Therefore, to provide a highly accurate biochemical evaluation of extracellular matrix using NIR Raman spectroscopy, freeze‐thaw processes are more suitable for ex vivo samples of human cartilage than formalin fixation. Copyright © 2015 John Wiley & Sons, Ltd.     

A shifted‐excitation Raman difference spectroscopy (SERDS) evaluation strategy for the efficient isolation of Raman spectra from extreme fluorescence interference

A biochemical characterization of pathologies in biological tissue can be provided by Raman spectroscopy. Often, the raw spectrum is severely affected by fluorescence interference. We report and compare various spectra‐processing approaches required for the purification of Raman spectra from heavily fluorescence‐interfered raw spectra according to the shifted‐excitation Raman difference spectroscopy method. These approaches cover the entire spectra‐processing chain from the raw spectra to the purified Raman spectra. In detail, we compared (1) area normalization versus z‐score normalization, (2) direct reconstruction of the difference spectra versus reconstruction of zero‐centered difference spectra and (3) collective baseline correction of the reconstructed spectra versus piecewise baseline correction of the reconstructed spectra and, finally, (4) analyzed the influence of the shift of the excitation wavelength on the quality of the reconstructed spectra. Statistical analysis of the spectra showed that – in our experiments – the best results were obtained for the z‐score normalization before subtraction of the normalized spectra, followed by zero‐centering of the difference spectra before reconstruction and a piecewise baseline correction of the pure Raman spectra. With our equipment, a wavelength shift from 784 to 785 nm provided reconstructed spectra of best quality. The analyzed specimens were different tissue types of pigs, tissue from the oral cavity of humans and a model solution of dye dissolved in ethanol. © 2015 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons Ltd.     

Study of both fingerprint and high wavenumber Raman spectroscopy of pathological nasopharyngeal tissues

High wavenumber (HW) Raman spectroscopy has weaker fluorescence background compared with fingerprint (FP) region. This study aims to evaluate the discrimination feasibility of nasopharyngeal non‐cancerous and nasopharyngeal cancer (NPC) tissue with both FP and HW Raman spectroscopy. HW Raman spectra of nasopharyngeal tissue were obtained for the first time. Raman spectra were collected to differentiate nasopharyngeal non‐cancerous (n = 37) from NPC (n = 41) tissues in FP (800–1800cm⁻¹), HW (2700–3100cm⁻¹), and integrated FP/HW region. First, to assess the utility of this method, the averaged Raman spectral intensities and intensity ratios of corresponding Raman bands were analyzed in HW and FP regions, respectively. The results show that intensities as well as the ratios of specific Raman peaks might be helpful in distinguishing nasopharyngeal non‐cancerous from NPC tissue with the HW Raman spectroscopy, as with FP Raman reported before. The multivariate statistical method based on the combination of principal component analysis–liner discriminant analysis (PCA‐LDA), together with leave‐one‐patient‐out, cross‐validation diagnostic algorithm, was used for discriminating nasopharyngeal non‐cancerous from NPC tissue, generating sensitivities of 87.8%, 85.4%, and 95.1% and specificities of 86.5%, 91.9%, and 89.2%, respectively, with Raman spectroscopy in the FP, HW, and integrated FP/HW regions. The posterior probability of classification results and receiver operating characteristic curves were utilized to evaluate the discrimination of PCA‐LDA algorithm, verifying that HW Raman spectroscopy has a positive effect on the differentiation for the diagnosis of NPC tissue by integrated FP/HW Raman spectroscopy. What's more, the potential of Raman spectroscopy used for differentiating different pathology NPC tissues was also discussed. The results demonstrate that both FP and HW Raman spectroscopy have the potential for diagnosis and detection in early nasopharyngeal carcinoma, and HW Raman spectroscopy may improve the discrimination of NPC tissue compared with FP region alone, providing a promising diagnostic tool for the diagnosis of NPC tissue. Copyright © 2015 John Wiley & Sons, Ltd.     

Diagnosis of head and neck squamous cell carcinoma using Raman spectroscopy: tongue tissues

Raman spectroscopy is a powerful optical technique capable of providing the structural information at the molecular level. Thus, the technique can be used to detect biochemical changes associated with carcinogenesis and identify the biomolecules involved in cancer. We studied the Raman spectral characteristics of normal, carcinoma in situ, and invasive squamous cell carcinoma (SCC) tissues of tongue, and identified the spectral features that can discriminate these three tissue types. We found that the intensities of Raman bands assignable to tryptophan increase while those attributable to protein keratin decrease when tissue changes from normal to invasive SCC. The variation observed in the intensity of many discriminating peaks including those of tryptophan and keratin as tissue changes from normal to carcinoma in situ and then to invasive SCC suggests that Raman spectroscopy can be used to monitor progression of the disease. We have also analyzed the data with multivariate statistical methods such as principal component analysis and discriminant function analysis. These chemometric methods clearly separate the whole data into three distinct groups consistent with results of pathology. We were able to detect with 91% success rate the normal and carcinoma in situ tissues and with 89% accuracy the invasive SCC tissues of the tongue. Copyright © 2011 John Wiley & Sons, Ltd.     

Near‐infrared Raman spectroscopy for gastric precancer diagnosis

Raman spectroscopy is a molecular vibrational spectroscopic technique that is capable of optically probing the biomolecular changes associated with neoplastic transformation. The purpose of this study was to apply near‐infrared (NIR) Raman spectroscopy for differentiating dysplasia from normal gastric mucosa tissue. A total of 65 gastric mucosa tissues (44 normal and 21 dysplasia) were obtained from 35 patients who underwent endoscopy investigation or gastrectomy operation for this study. A rapid NIR Raman system was utilized for tissue Raman spectroscopic measurements at 785‐nm laser excitation. High‐quality Raman spectra in the range of 800–1800 cm⁻¹ can be acquired from gastric mucosa tissue within 5 s. Raman spectra showed significant differences between normal and dysplastic tissue, particularly in the spectral ranges of 850–1150, 1200–1500 and 1600–1750 cm⁻¹, which contained signals related to proteins, nucleic acids and lipids. The diagnostic decision algorithm based on the combination of Raman peak intensity ratios of I₈₇₅/I₁₄₅₀ and I₁₂₀₈/I₁₆₅₅ and the logistic regression analysis yielded a diagnostic sensitivity of 90.5% and specificity of 90.9% for identification of gastric dysplasia tissue. This work demonstrates that NIR Raman spectroscopy in conjunction with intensity ratio algorithms has the potential for the noninvasive diagnosis and detection of precancer in the stomach at the molecular level. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

SERS‐based detection of barcoded gold nanoparticle assemblies from within animal tissue

We have explored the potential of deep Raman spectroscopy, specifically surface‐enhanced spatially offset Raman spectroscopy (SESORS), for non‐invasive detection from within animal tissue, by employing SERS‐barcoded nanoparticle (NP) assemblies as the diagnostic agent. This concept has been experimentally verified in a clinically‐relevant backscattered Raman system with an excitation line of 785 nm under ex vivo conditions. We have shown that our SORS system, with a fixed offset of 2–3 mm, offered sensitive probing of injected 2‐quinolinethiol‐barcoded NP assemblies through animal tissue containing both protein and lipid. In comparison with that of non‐aggregated SERS‐barcoded gold NPs, we have demonstrated that the tailored SERS‐barcoded aggregated NP assemblies have significantly higher detection sensitivity. We report that these NP assemblies can be readily detected at depths of 7–8 mm from within animal proteinaceous tissue with high signal‐to‐noise ratio. In addition, they could also be detected from beneath 1–2 mm of animal tissue with high lipid content, which generally poses a challenge because of high absorption of lipids in the near‐infrared region. We have also shown that the signal intensity and signal‐to‐noise ratio at a particular depth is a function of the SERS tag concentration used and that our SORS system has a 2‐quinolinethiol detection limit of 10⁻⁶ M. Higher detection depths may possibly be obtained with optimization of the NP assemblies, along with improvements in the instrumentation. Such NP assemblies offer prospects for in vivo, non‐invasive detection of tumours along with scope for incorporation of drugs and their targeted and controlled release at tumour sites. These diagnostic agents combined with drug delivery systems could serve as a ‘theranostic agent’, an integration of diagnostics and therapeutics into a single platform. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of epidermal L‐tryptophan and its oxidation products by in vivo FT‐Raman Spectroscopy further supports oxidative stress in patients with vitiligo

In the past, non‐invasive in vivo FT‐Raman spectroscopy has been used to detect H₂O₂‐mediated oxidation of methionine to methionine sulfoxide and methionine sulfone, as well as cysteine to cysteic acid, in the sequence of proteins in the epidermis of patients with vitiligo. L ‐tryptophan (Trp) is another potential target for this oxidation. Owing to the presence of 10⁻³M epidermal albumin which contains one Trp residue, it was tempting to follow the oxidation of this amino acid. Using in vivo and in vitro FT‐Raman spectroscopy, we show for the first time that epidermal Trp is oxidised in patients with vitiligo, yielding 5‐OH‐Trp at 930 cm⁻¹ and other oxidation products (i.e. N‐formyl kynurenine and kynurenine) from indole ring oxidation peaking at 1050 cm⁻¹. On the basis of detailed in vitro results, we could conclude that 5‐OH‐Trp as well as formyl kynurenine and kynurenine are formed via H₂O₂‐mediated Fenton chemistry. These results once again bring out the strength of non‐invasive in vivo FT‐Raman Spectroscopy in dermatology to follow the effect of oxidative stress in the skin of patients with vitiligo. Copyright © 2008 John Wiley & Sons, Ltd.     

Photoacoustic microscopy

Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (∼1 mm in soft tissue). With its excellent scalability, PAM can provide high‐resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering‐based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence‐based methods, such as wide‐field, confocal, and multi‐photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state‐of‐the‐art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies.     

Assessment of ex vivo perfused liver health by Raman spectroscopy

Raman spectroscopy was applied in this research to monitor the overall health and degradation of porcine livers perfused ex vivo using the VasoWaveW® perfusion system. A novel Raman‐based diagnostic analysis was developed that enables near real‐time and label‐free monitoring of organ health during ex vivo perfusion designed to extend the useful life of the organ for transplantation. Multivariate statistical analysis of Raman spectra of organ perfusate fluid, using a combination of principal component analysis and linear discriminant analysis, proved to be an effective technique to assess the degradation properties of the livers. Three livers (with replicates) were perfused ex vivo under different pressures and temperatures and were compared with a 24‐h time‐course. Results indicated that perfusion pressure was a more significant factor in organ degradation than was temperature. In addition, a non‐linear degradation profile was identified for all three perfused livers, and this profile was different for individual livers, demonstrating the time‐dependent transition from its initial ‘healthy’ state towards a more ‘unhealthy’ degenerative state at 24 h. The Raman spectroscopy‐based approach described here has potential applications in perfusion and diagnostic instrumentation that can be used in near real‐time during organ transit and in operating rooms to help identify appropriately healthy organs for transplantation. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of laser excitation wavelength and optical mode on Raman spectra of human fresh colon,pancreas, and prostate tissues

Early cancer detection is the central and most important factor for allowing successful treatment and resultant positive long‐term patient outcomes. Recently, optical techniques have been applied to this purpose, although each has inherent limitations. In particular, Raman spectroscopy applied in the pathological diagnosis of cancerous tissues has received increasing attention, with the merit of being highly sensitive to the biochemical alterations in tissue compositions and applicable in vivo. Nevertheless, its application has been impeded by the high background intensity, which masks the Raman signal of biological molecules. In this work, the influence of laser excitation wavelength (785 vs. 830 nm) and optical mode (single mode vs. multimode) on the background intensity of fresh human tissues was studied. Based on the results, laser with 830 nm excitation demonstrated better background reduction than that with 785 nm excitation for the same optical mode, but the Raman signal intensity was conversely reduced, and the signal‐to‐noise ratio (SNR) not improved. In contrast, by comparing single‐mode and multimode 785 nm excitations, it was shown that the single‐mode laser with its smaller beam waist and beam propagation factor had better background reduction ability and an improvement of the SNRs. It is speculated that this decrease in background intensity comes from the effect of the optical mode on the Mie scattering from the biological tissue. High‐quality spectra based on a careful selection of both laser excitation wavelength and optical mode will benefit Raman measurements in further research focusing on spectral interpretation and histopathological correlation ultimately aimed toward intraoperative applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Dipole‐like backscatter stimulated Raman scattering for in vivo imaging

Stimulated Raman scattering (SRS) scanning microscopy has the potential to enable label‐free in vivo imaging for research and clinical medicine. Volume SRS from focus occurs in the forward scattered direction. Therefore, multiple scattering events are required to direct the light out of the tissue, reducing imaging depth and resolution. Here, a method called Stokes interference SRS (SISRS) is introduced that operates by the addition to the standard pump and stimulated emission probe beams a third beam called the donut beam. The donut is close in wavelength to the probe beam and, after passage through a π phase plate, forms an annular beam in the focal plane with bright nodes above and below focus. The donut beats with the probe beam, and when they destructively interfere with each other, the microscope's 3‐D stimulated emission focal spot is reduced to subwavelength dimensions. A subwavelength focal volume emits a dipole pattern of SRS with forward and backscatter lobes, enabling high‐resolution single‐backscatter imaging from deep within tissues. The reduction of the focal volume also increases the resolution of the scanning image creating imaging beyond the diffraction limit. SISRS imaging may provide in vivo label‐free Raman images comparable with that achieved in stained in vitro tissues in all planes of section. Copyright © 2014 John Wiley & Sons, Ltd.