Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells

Noninvasive monitoring of tissue-engineered (TE) constructs during their in vitro maturation or postimplantation in vivo is highly relevant for graft evaluation. However, traditional methods for studying cell and matrix components in engineered tissues such as histology, immunohistochemistry, or biochemistry require invasive tissue processing, resulting in the need to sacrifice of TE constructs. Raman spectroscopy offers the unique possibility to analyze living cells label-free in situ and in vivo solely based on their phenotype-specific biochemical fingerprint. In this study, we aimed to determine the applicability of Raman spectroscopy for the noninvasive identification and spectral separation of primary human skin fibroblasts, keratinocytes, and melanocytes, as well as immortalized keratinocytes (HaCaT cells). Multivariate analysis of cell-type-specific Raman spectra enabled the discrimination between living primary and immortalized keratinocytes. We further noninvasively distinguished between fibroblasts, keratinocytes, and melanocytes. Our findings are especially relevant for the engineering of in vitro skin models and for the production of artificial skin, where both the biopsy and the transplant consist of several cell types. To realize a reproducible quality of TE skin, the determination of the purity of the cell populations as well as the detection of potential molecular changes are important. We conclude therefore that Raman spectroscopy is a suitable tool for the noninvasive in situ quality control of cells used in skin tissue engineering applications.     

Prediction of glucose in whole blood by near-infrared spectroscopy: influence of wavelength region, preprocessing, and hemoglobin concentration

Measurement accuracy for predicting glucose in whole blood was studied based on near-infrared spectroscopy. Optimal wavelength regions, preprocessing, and the influence of hemoglobin were examined using partial least-squares regression. Spectra between 1100 and 2400 nm were measured from 98 whole blood samples. In order to study the influence of hemoglobin, which is the most dominant component in blood, 98 samples were arranged such that glucose and hemoglobin concentrations were distributed in their physiological ranges. Samples were grouped into three depending on hemoglobin level. The results showed that glucose prediction was influenced by hemoglobin concentrations in the calibration model. It was necessary for samples used in the calibration model to represent the entire range of hemoglobin level. The cross-validation errors were the smallest when the wavelength regions of 1390 to 1888 nm and 2044 to 2393 nm were used. However, prediction accuracy was not very dependent on preprocessing methods in this optimal region. The standard error of glucose prediction was 25.5 mgdL and the coefficient of variation in prediction was 11.2%.     

Specificity of noninvasive blood glucose sensing using optical coherence tomography technique: a pilot study

Noninvasive monitoring of blood glucose concentration in diabetic patients would significantly reduce complications and mortality associated with this disease. In this paper, we experimentally and theoretically studied specificity of noninvasive blood glucose monitoring with the optical coherence tomography (OCT) technique. OCT images and signals were obtained from skin of Yucatan micropigs and New Zealand rabbits. Obtained results demonstrate that: (1) several body osmolytes may change the refractive index mismatch between the interstitial fluid (ISF) and scattering centres in tissue, however the effect of the glucose is approximately one to two orders of magnitude higher; (2) an increase of the ISF glucose concentration in the physiological range (3-30 mM) may decrease the scattering coefficient by 0.22% mM(-1) due to cell volume change; (3) stability of the OCT signal slope is dependent on tissue heterogeneity and motion artefacts; and (4) moderate skin temperature fluctuations (+/- 1 degree C) do not decrease accuracy and specificity of the OCT-based glucose sensor, however substantial skin heating or cooling (several degrees C) significantly change the OCT signal slope. These results suggest that the OCT technique may provide blood glucose concentration monitoring with sufficient specificity under normal physiological conditions.     

Raman spectroscopy for diagnosis of atherosclerosis: a rapid analysis using neural networks

Near-infrared Raman spectroscopy (NIRS) is one of the novel techniques that has a potential for in vivo diagnosis of atherosclerosis in human arteries. For such real time clinical applications, a rapid collection and analysis of the data is needed. One of the major problems with the fast data collection is that the noise generated by the detector has the same level as the Raman signal from the tissue, which makes the analysis difficult. In this work, NIRS measurements have been carried out on a total of 60 samples from human coronary arteries. Raman spectral data with the correlated histopathological analysis have been used as a basis to stimulate the cases of severe noise conditions. The main objective of this paper is the comparison of different processing algorithms that have been developed based on either wavelet transformation or principal component analysis for compressing the Raman spectral vectors and a rapid data classification based on different neural network architectures. The developed algorithms found to provide promising diagnosis results with classification errors smaller than 5%, even in the cases of Raman data with collection times as small as 20 ms. It has been concluded that the developed algorithms would be very much useful in the development of Raman spectroscopy systems for in vivo biological applications.     

In vivo quantification of photosensitizer concentration using fluorescence differential path-length spectroscopy: influence of photosensitizer formulation and tissue location

In vivo measurement of photosensitizer concentrations may optimize clinical photodynamic therapy (PDT). Fluorescence differential path-length spectroscopy (FDPS) is a non-invasive optical technique that has been shown to accurately quantify the concentration of Foscan? in rat liver. As a next step towards clinical translation, the effect of two liposomal formulations of mTHPC, Fospeg? and Foslip?, on FDPS response was investigated. Furthermore, FDPS was evaluated in target organs for head-and-neck PDT. Fifty-four healthy rats were intravenously injected with one of the three formulations of mTHPC at 0.15 mg kg(-1). FDPS was performed on liver, tongue, and lip. The mTHPC concentrations estimated using FDPS were correlated with the results of the subsequent harvested and chemically extracted organs. An excellent goodness of fit (R(2)) between FDPS and extraction was found for all formulations in the liver (R(2)=0.79). A much lower R(2) between FDPS and extraction was found in lip (R(2)=0.46) and tongue (R(2)=0.10). The lower performance in lip and in particular tongue was mainly attributed to the more layered anatomical structure, which influences scattering properties and photosensitizer distribution.     

Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using a precision open-ended coaxial probe

Hepatic malignancies have historically been treated with surgical resection. Due to the shortcomings of this technique, there is interest in other, less invasive, treatment modalities, such as microwave hepatic ablation. Crucial to the development of this technique is the accurate knowledge of the dielectric properties of human liver tissue at microwave frequencies. To this end, we characterized the dielectric properties of in vivo and ex vivo normal, malignant and cirrhotic human liver tissues from 0.5 to 20 GHz. Analysis of our data at 915 MHz and 2.45 GHz indicates that the dielectric properties of ex vivo malignant liver tissue are 19 to 30% higher than normal tissue. The differences in the dielectric properties of in vivo malignant and normal liver tissue are not statistically significant (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 16% higher than normal). Also, the dielectric properties of in vivo normal liver tissue at 915 MHz and 2.45 GHz are 16 to 43% higher than ex vivo. No statistically significant differences were found between the dielectric properties of in vivo and ex vivo malignant tissue (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 28% higher than normal). We report the one-pole Cole-Cole parameters for ex vivo normal, malignant and cirrhotic liver tissue in this frequency range. We observe that wideband dielectric properties of in vivo liver tissue are different from the wideband dielectric properties of ex vivo liver tissue, and that the in vivo data cannot be represented in terms of a Cole-Cole model. Further work is needed to uncover the mechanisms responsible for the observed wideband trends in the in vivo liver data.     

Effect of marker cluster design on the accuracy of human movement analysis using stereophotogrammetry

This paper presents a new, simple model to evaluate the instrumental random errors in kinematic analysis of human movements using stereophotogrammetry. By means of equations analogous to that relate linear or angular momentum with linear or angular velocities, a direct measurement of instantaneous motion can be made without previous finite displacement analysis. Single explicit expressions can be obtained to evaluate the influence of instrumental random errors in the accuracy of the kinematic variables. From these expressions, some conclusions about the effect of marker cluster design on the experimental errors are obtained. An experiment has been carried out in order to validate the proposed technique and to assess the experimental errors in linear and angular velocity measurement and its influence in instantaneous helical axis determination.     

复方茯苓制剂对营养性肥胖大鼠体重、血糖、血脂及小肠肠系膜微循环的影响

目的：观察复方茯苓制剂(CPP)对肥胖大鼠体重、血流动力学、血糖、血脂、小肠肠系膜微循环的影响,探讨防治肥胖症的新途径。 方法： Wistar大鼠45只分为普通饲料喂养组（A组）、高能饲料喂养组（B组）、高能量饲料喂养+复方茯苓制剂组（C组），分别观测体重、血压、右心房压、血糖、血脂及肠系膜微循环的变化。 结果： B组用CPP治疗后平均体重由(313.00±17.29)g降至(217.50±17.50)g（P＜0.01);体动脉平均血压由(173.88±2.97)mmHg降至(101.73±3.35)mmHg（P<0.01）,右房平均压从（13.58±3.59）mmHg下降为（11.32±0.68）mmHg(P<0.05);大鼠肠系膜毛细血管管径由（7.93±0.90）μm降为（3.93±0.90）μm(P<0.05)；血流速度从（270.92±49.73）μm/s增至（410.13±76.54）μm/s（P<0.01）；血浆极低密度脂蛋白（VLDL）由（3.18±0.01）mmol/L增加至（4.55±0.01）mmol/L;总胆固醇（T-Chol）从（7.87±0.01）mmol/L降至（5.56±0.01）mmol/L（P<0.05），血糖由（12.87±0.04）mmol/L下降至（8.97±0.07）mmol/L(P<0.05)。上述指标参数与普通饲料喂养组相比无显著差异(P>0.05)。 结论： 复方茯苓制剂能使肥胖大鼠减肥及改善小肠肠系膜微循环。     

Pilot study for detection of early changes in tissue associated with heterotopic ossification: moving toward clinical use of Raman spectroscopy

Abstract

Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions (?<?16 d post-injury), early HO lesions (?>?16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (p?<?0.01) and the 1340/1270?cm^?1 (p?<?0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.     

Immunohistochemical analysis of adenocarcinoma of the small intestine: a tissue microarray study

BACKGROUND: Primary adenocarcinomas of the small intestine are rare, and the genetic mechanisms involved in their carcinogenesis remain unclear. AIM: To examine the expression of candidate proteins in small intestinal adenocarcinomas by immunohistochemistry performed on tissue microarrays (TMAs). METHODS: Twenty seven primary sporadic small intestinal adenocarcinomas were analysed. The TMA technique was validated by comparing immunohistochemical labelling of hMLH1 and hMSH2 on TMAs and the tissue sections they derived from. The expression of Smad4, hMSH6, beta catenin, and p53 was investigated and results compared with those obtained in 14 malignant ampullary tumours. RESULTS: TMA technology with threefold redundancy adequately represented the immunohistochemical pattern of small intestinal adenocarcinomas. Loss of hMLH1 expression, but not hMSH2 or hMSH6, was seen in two of 27 small intestinal adenocarcinomas. All ampullary tumours showed nuclear staining for hMSH2 and hMSH6. One case showed lack of immunostaining for hMLH1. Smad4 expression was absent in five small intestinal adenocarcinomas and two ampullary tumours. Overexpression of p53 was detected in the nuclei of 14 of the 27 small intestinal adenocarcinomas, and five of the 14 ampullary tumours. Nuclear or cytoplasmic expression of beta catenin was present in all specimens. CONCLUSION: Inactivation of the SMAD4/DPC4 gene seems to be involved in small intestinal adenocarcinoma tumorigenesis. Overexpression of p53 and abnormal expression of beta catenin are two common events, unlike the loss of expression of the DNA mismatch repair proteins (hMLH1, hMSH2, and hMSH6). The carcinogenetic process appears to be similar in small intestinal adenocarcinomas and malignant ampullary tumours.     

Quantitative spatially resolved measurement of tissue chromophore concentrations using photoacoustic spectroscopy: application to the measurement of blood oxygenation and haemoglobin concentration

A new approach based on pulsed photoacoustic spectroscopy for non-invasively quantifying tissue chromophore concentrations with high spatial resolution has been developed. The technique is applicable to the quantification of tissue chromophores such as oxyhaemoglobin (HbO(2)) and deoxyhaemoglobin (HHb) for the measurement of physiological parameters such as blood oxygen saturation (SO(2)) and total haemoglobin concentration. It can also be used to quantify the local accumulation of targeted contrast agents used in photoacoustic molecular imaging. The technique employs a model-based inversion scheme to recover the chromophore concentrations from photoacoustic measurements. This comprises a numerical forward model of the detected time-dependent photoacoustic signal that incorporates a multiwavelength diffusion-based finite element light propagation model to describe the light transport and a time-domain acoustic model to describe the generation, propagation and detection of the photoacoustic wave. The forward model is then inverted by iteratively fitting it to measurements of photoacoustic signals acquired at different wavelengths to recover the chromophore concentrations. To validate this approach, photoacoustic signals were generated in a tissue phantom using nanosecond laser pulses between 740 nm and 1040 nm. The tissue phantom comprised a suspension of intralipid, blood and a near-infrared dye in which three tubes were immersed. Blood at physiological haemoglobin concentrations and oxygen saturation levels ranging from 2% to 100% was circulated through the tubes. The signal amplitude from different temporal sections of the detected photoacoustic waveforms was plotted as a function of wavelength and the forward model fitted to these data to recover the concentrations of HbO(2) and HHb, total haemoglobin concentration and SO(2). The performance was found to compare favourably to that of a laboratory CO-oximeter with measurement resolutions of +/-3.8 g l(-1) (+/-58 microM) and +/-4.4 g l(-1) (+/-68 microM) for the HbO(2) and HHb concentrations respectively and +/-4% for SO(2) with an accuracy in the latter in the range -6%-+7%.     

Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm(-1)) and a decrease in reducible cross-links (1690 cm(-1)). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.     

Noninvasive evaluation of collagen and hemoglobin contents and scattering property of in vivo keloid scars and normal skin using diffuse reflectance spectroscopy: pilot study

Collagen is a rich component in skin that provides skin structure integrity; however, its contribution to the absorption and scattering properties of various types of skin has not been extensively studied. We considered the contribution of the collagen to the absorption spectrum of in vivo normal skin and keloids of 12 subjects derived from our diffuse reflectance spectroscopy (DRS) system in the wavelength range from 550 to 860 nm. It was found that the collagen concentration, the hemoglobin oxygen saturation, and the reduced scattering coefficient of keloids were remarkably different from that of normal skin. Our results suggest that our DRS system could assist clinicians in understanding the functional and structural condition of keloid scars. In the future, we will evaluate the accuracy of our system in the keloid diagnosis and investigate the applicability of our system for other skin-collagen-related studies.     

Habits with killer instincts: in vivo analysis on the severity of oral mucosal alterations using autofluorescence spectroscopy

Oral habits like chewing and smoking are main causes of oral cancer, which has a higher mortality rate than many other cancer forms. Currently, the long term survival rate of oral cancer is less than 50%, as a majority of cases are detected very late. The clinician's main challenge is to differentiate among a multitude of red, white, or ulcerated lesions. Hence, new noninvasive, reliable, and fast techniques for the discrimination of oral cavity disorders are to be developed. This study includes autofluorescence spectroscopic screening of normal volunteers with and without lifestyle oral habits and patients with oral submucous fibrosis (OSF). The spectra from different sites of habitue?s, non-habitue?s, and OSF patients were analyzed using the intensity ratio, redox ratio, and linear discriminant analysis (LDA). The spectral disparities among these groups are well demonstrated in the emission regions of collagen and Flavin adenine dinucleotide. We observed that LDA gives better efficiency of classification than the intensity ratio technique. Even the differentiation of habitue?s and non-habitue?s could be well established with LDA. The study concludes that the clinical application of autofluorescence spectroscopy along with LDA, yields spontaneous screening among individuals, facilitating better patient management for clinicians and better quality of life for patients.     

A semi-automated method using interpolation and optimisation for the 3D reconstruction of the spine from bi-planar radiography: a precision and accuracy study

The 3D reconstruction of the spine in upright posture can be obtained by bi-planar radiographic methods, developed since the 1970s. The principle is to identify 4–25 anatomical landmarks per vertebrae and per images. This identification time is hardly manageable in clinical practice. A semi-automated method is used: 3D standard vertebral models are positioned along with a 3D curve (identified all the way through the vertebral bodies). The silhouettes of the models of C7 and L5 vertebrae are first adjusted and the positions of the other vertebrae are interpolated and optimised. The inter- and intra-operator variabilities and the errors between the semi-automated method and the manual identification of six anatomical landmarks per vertebra are evaluated on 20 pairs of X-ray images of subjects with different spinal deformities. The identification time for the semi-automated method is 5 min. For scolitic subjects, the precision is under 2.2° and the accuracy is under 3.2° for all lateral, sagittal and axial rotations.