Stokes shift spectroscopy pilot study for cancerous and normal prostate tissues

Stokes shift spectroscopy (S3) is an emerging approach toward cancer detection. The goal of this paper is to evaluate the diagnostic potential of the S3 technique for the detection and characterization of normal and cancerous prostate tissues. Pairs of cancerous and normal prostate tissue samples were taken from each of eight patients. Stokes shift spectra were measured by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval Δλ=20 nm between them. The salient features of this technique are the highly resolved emission peaks and significant spectral differences between the normal and cancerous prostate tissues, as observed in the wavelength region of 250 to 600 nm. The Stokes shift spectra of cancerous and normal prostate tissues revealed distinct peaks around 300, 345, 440, and 510 nm, which are attributed to tryptophan, collagen, NADH, and flavin, respectively. To quantify the spectral differences between the normal and cancerous prostate tissues, two spectral ratios were computed. The findings revealed that both ratio parameters R1=I297/I345 and R2=I307/I345 were excellent diagnostic ratio parameters giving 100% specificity and 100% sensitivity for distinguishing cancerous tissue from the normal tissue. Our results demonstrate that S3 is a sensitive and specific technique for detecting cancerous prostate tissue.     

Characteristic spectral features of the polarized fluorescence of human breast cancer in the wavelet domain

Wavelet transform of polarized fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate normal and malignant tissue types. The intensity differences of parallel and perpendicularly polarized fluorescence spectra are subjected to investigation, since they are relatively free of diffusive background. A number of parameters, capturing spectral variations and subtle changes in the diseased tissues in the visible wavelength regime, are clearly identifiable in the wavelet domain. These manifest both in the average low-pass and high frequency high-pass wavelet coefficients.     

Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer

Gold nanoparticles with unique optical properties may be useful as biosensors in living whole cells. Using a simple and inexpensive technique, we recorded surface plasmon resonance (SPR) scattering images and SPR absorption spectra from both colloidal gold nanoparticles and from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies after incubation in cell cultures with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). Colloidal gold nanoparticles are found in dispersed and aggregated forms within the cell cytoplasm and provide anatomic labeling information, but their uptake is nonspecific for malignant cells. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells. These results suggest that SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.     

Influence of mineral components on laser-induced fluorescence spectra of calcified human heart-valve tissues

Laser-induced fluorescence (LIF) spectra of calcified human heart-valve tissue and LIF spectra of macroscopic calcinosis fragments dissected from human heart valves were compared with LIF spectra of pig myocardium tissues. Excitation was provided by an excimer laser with wavelength lambda = 248 nm. Fluorescence bands that were due to mineral and organic tissue components were identified by measurement of LIF spectra of macroscopic fragments of calcified tissues that had been heat treated at 700 degrees C. The studies showed that LIF spectra of calcified tissues include fluorescence emission from tryptophan, collagen, elastin, and a mineral component of tissue, hydroxylapatite. The observed differences in LIF spectra of normal and calcified tissues with different pathologies may result not only from calcification-induced changes in relative collagen and elastin concentrations but also from additional (absent in normal heart tissue) fluorescence of hydroxylapatite. The calcification-induced changes in the LIF spectra of human heart-valve tissues, characterized by a 330/450 nm ratio, were found to be quite appreciable, which suggests that this ratio can be used with LIF measurements to evaluate the degree of heart-tissue calcification.     

Monte Carlo-based inverse model for calculating tissue optical properties. Part II: Application to breast cancer diagnosis

The Monte Carlo-based inverse model of diffuse reflectance described in part I of this pair of companion papers was applied to the diffuse reflectance spectra of a set of 17 malignant and 24 normal-benign ex vivo human breast tissue samples. This model allows extraction of physically meaningful tissue parameters, which include the concentration of absorbers and the size and density of scatterers present in tissue. It was assumed that intrinsic absorption could be attributed to oxygenated and deoxygenated hemoglobin and beta-carotene, that scattering could be modeled by spheres of a uniform size distribution, and that the refractive indices of the spheres and the surrounding medium are known. The tissue diffuse reflectance spectra were evaluated over a wavelength range of 400-600 nm. The extracted parameters that showed the statistically most significant differences between malignant and nonmalignant breast tissues were hemoglobin saturation and the mean reduced scattering coefficient. Malignant tissues showed decreased hemoglobin saturation and an increased mean reduced scattering coefficient compared with nonmalignant tissues. A support vector machine classification algorithm was then used to classify a sample as malignant or nonmalignant based on these two extracted parameters and produced a cross-validated sensitivity and specificity of 82% and 92%, respectively.     

Autofluorescence and diffuse reflectance spectroscopy of oral epithelial tissue using a depth-sensitive fiber-optic probe

Optical spectroscopy can provide useful diagnostic information about the morphological and biochemical changes related to the progression of precancer in epithelial tissue. As precancerous lesions develop, the optical properties of both the superficial epithelium and underlying stroma are altered; measuring spectral data as a function of depth has the potential to improve diagnostic performance. We describe a clinical spectroscopy system with a depth-sensitive, ball lens coupled fiber-optic probe for noninvasive in vivo measurement of oral autofluorescence and diffuse reflectance spectra. We report results of spectroscopic measurements from oral sites in normal volunteers and in patients with neoplastic lesions of the oral mucosa; results indicate that the addition of depth selectivity can enhance the detection of optical changes associated with precancer.     

Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject

A 1-GHz multifrequency, multiwavelength frequency-domain photon migration instrument is used to measure quantitatively the optical absorption (mu(a)) and effective optical scattering (mu(s) ?) of normal and malignant tissues in a human subject. Large ellipsoidal (~10-cm major axis, ~6-cm minor axes) subcutaneous malignant lesions were compared with adjacent normal sites in the abdomen and back. Absorption coefficients recorded at 674, 811, 849, and 956 nm were used to calculate tissue hemoglobin concentration (oxyhemoglobin, deoxyhemoglobin, and total), water concentration, hemoglobin oxygen saturation, and blood volume fraction in vivo. Our results show that the normal and the malignant tissues measured in the patient have clearly resolvable optical and physiological property differences that may be broadly useful in identifying and characterizing tumors.     

乳腺癌中着丝粒蛋白CenpG的差异表达

用间接免疫荧光（IIF）验证了着丝粒蛋白CenpG的一种抗血清；通过对31例乳腺癌组织块及每一例病人的癌旁正常（非癌性）组织块蛋白提取物进行免疫印迹（Western blot）分析，发现多数情况下（71%）癌组织中着丝粒蛋白CenpG(95kD)过表达；在两例乳腺癌组织中还发现了一种能被CenpG抗血清识别的43kD蛋白质成分。结果表明，新近发现和命名的CenpG（或者还有其他相关成分）的差异表达可能与细胞恶性增殖有关。     

Noninvasive subsurface analysis using multiple miniaturized Raman probes, part I: basic study of thin-layered transparent models of biomedical tissues

This study describes a basic theory for reconstructing pure Raman signals of materials composing a multilayer sample from Raman spectra obtained using two types of miniaturized Raman probes. An illustrative example is demonstrated using a multilayer system of samples composed of the transparent plastics polymethylmethacrylate (PMMA) and polyethylene (PE) as a model of thin-layered biomedical tissues. When the same region of an object is measured using Raman probes with different focal properties, the Raman spectra provide different depth profile information depending on the level of light penetration. Thus, a detailed comparison of the spectra can provide an interesting opportunity to probe the differences between the layers. A simple analytic form is presented for reconstructing the pure Raman spectra of the embedded layer. The method applies an understanding of the Raman sampling volume in layered transparent materials to the interpretation of Raman spectra experimentally measured by multiple probes. The basic theory described here is necessary for the expansion of the technique to turbid media, such as biological samples, where light-scattering effects must be considered. The potential applications of the proposed method include material and catalyst subsurface probing through different embedded materials, such as assessment of silicon wafers, effective noninvasive screening for catalyst synthesis, and biomedical tissue research.     

Raman-active modes of single-walled carbon nanotubes derived from the gas-phase decomposition of CO (HiPco process)

Here we report Raman scattering studies of ropes of Single-walled carbon nanotubes (SWNTs) grown by a high CO pressure process. Five samples from five different batches were studied as a function of excitation wavelength. Three of these samples exhibited Raman spectra similar to that found for SWNTs made by pulsed laser vaporization of arc-discharge methods. The other two samples were found by Raman scattering to contain a significant fraction of tubes with diameter < 1.0 nm. These samples exhibited unusual spectra that, however, can be well understood within the existing models for the electronic and phononic states in SWNTs. Spectra recorded with 1064 nm for the sample having a significant fraction of smaller diameter tubes shows strong modes present between 500 and 1200 cm-1. We suggest these modes arise due to the enhancement of Raman cross-section for small diameter tubes.     

Determination of optical coefficients and fractal dimensional parameters of cancerous and normal prostate tissues

Optical extinction and diffuse reflection spectra of cancerous and normal prostate tissues in the 750 to 860 nm spectral range were measured. Optical extinction measurements using thin ex vivo prostate tissue samples were used to determine the scattering coefficient (μ(s)), while diffuse reflection measurements using thick prostate tissue samples were used to extract the absorption coefficient (μ(a)) and the reduced scattering coefficient (μ'(s)). The anisotropy factor (g) was obtained using the extracted values of μ(s) and μ'(s). The values of fractal dimension (D(f)) of cancerous and normal prostate tissues were obtained by fitting to the wavelength dependence of μ'(s). The number of scattering particles contributing to μ(s) as a function of particle size and the cutoff diameter d(max) as a function of g were investigated using the fractal soft tissue model and Mie theory. Results show that d(max) of the normal tissue is larger than that of the cancerous tissue. The cutoff diameter d(max) is observed to agree with the nuclear size for the normal tissues and the nucleolar size for the cancerous tissues. Transmission spectral polarization imaging measurements were performed that could distinguish the cancerous prostate tissue samples from the normal tissue samples based on the differences between their absorption and scattering parameters.     

Far-infrared spectroscopy of protein higher-order structures

Far-infrared (FIR) spectroscopy in the spectral region of 50-450 cm(-1) was used to study a series of protein higher-order structures constructed using β-lactoglobulin and polyomavirus capsid protein VP1. There were marked differences in the spectra for β-lactoglobulin monomer and dimer and between untreated β-lactoglobulin and heat-induced gels formed at neutral pH. Untreated β-lactoglobulin and heat-induced gels formed at acidic pH exhibited little difference in their spectra. Assembly of the quaternary structure of polyomavirus virus-like particles also caused large changes in the FIR spectra. These findings suggest that FIR spectroscopy may prove useful in studying some protein quaternary and higher-order structures. There was evidence of detection of β-lactoglobulin dimerization, intermolecular disulfide bonding in heat-induced neutral gels, and polyomavirus virus-like particle assembly but no evidence that FIR could detect β-lactoglobulin fibrils with their polymeric structure and hydrogen-bonded intermolecular β-pleated sheeting.     

Determination of Elastic Properties of Resected Human Breast Tissue Samples Using Optical Coherence Tomographic Elastography

Abstract: The present study is concerned with the application of optical coherence tomographic (OCT) elastography technique for quantitative assessment of the elastic properties of resected human breast tissue samples subjected to axial compressive loading in vitro. Three classes of breast tissue samples, namely normal, benign (fibroadenoma) and malignant (invasive ductal carcinoma), were considered. A speckle tracking technique based on two‐dimensional cross correlation was employed to track the speckle motion between original (pre‐compressed) and the displaced (post‐compressed) OCT images of the tissue samples for the measurement of displacement and strain maps. The overall data reduction approach for quantitative assessment of elastic properties was validated against the results of gelatin phantoms containing activated charcoal particles as scattering centres. Results are presented in the form of OCT images and displacement and axial strain maps for normal, benign and malignant breast tissue samples. Based on the stress–strain relationship obtained for these three classes, the values of stiffness coefficients were reported in terms of modulus of elasticity. Results of the study reveal significant differences between the two‐dimensional displacement vector maps of normal and cancerous breast tissue samples. The stiffness of benign tissue samples is found to be about two times higher than that of normal tissue samples, whereas for malignant samples, it is about four times higher, thereby signifying appreciable differences in the stiffness of cancerous and normal tissue samples.     

Confocal Raman microscopy and SEM/EDS investigations of the interface between the zirconia core and veneering ceramic: the influence of a liner and regeneration firing

The aim of this study was to evaluate the changes in the transition layer at the interface between yttria partial stabilized tetragonal zirconia polycrystalline (Y-TZP) core and veneering feldspathic ceramic (VITA VM(?)9), under different manufacturing methods. Confocal Raman microscopy and energy dispersive X-ray spectroscopy (EDS) analyses were carried out on tapered veneered cross sections of the interface. For some samples, an additional firing of the core was used, as the application of an optional liner material between the core and veneer. Single Raman spectra were distinguishable between Y-TZP and the veneering materials. VM(?)9 and liner spectra were broadly superimposable. No substantial differences appeared in their chemical elemental composition. 2D Raman images and EDS analysis emphasized changes in the interdiffusion thickness; the additional firing of the core decreased the interdiffusion zone, and the highest firing temperature of the liner increased the interdiffusion zone. These results, which will help us understand the changes in this transition layer, are discussed.     

Development of a method for the determination of Fusarium fungi on corn using mid-infrared spectroscopy with attenuated total reflection and chemometrics

A novel method, which enables the determination of fungal infection with Fusarium graminearum on corn within minutes, is presented. The ground sample was sieved and the particle size fraction between >250 and 100 microm was used for mid-infrared/attenuated total reflection (ATR) measurements. The sample was pressed onto the ATR crystal, and reproducible pressure was applied. After the spectra were recorded, they were subjected to principle component analysis (PCA) and classified using cluster analysis. Observed changes in the spectra reflected changes in protein, carbohydrate, and lipid contents. Ergosterol (for the total fungal biomass) and the toxin deoxynivalenol (DON; a secondary metabolite) of Fusarium fungi served as reference parameters, because of their relevance for the examination of corn based food and feed. The repeatability was highly improved by sieving prior to recording the spectra, resulting in a better clustering in PCA score/score plots. The developed method enabled the separation of samples with a toxin content of as low as 310 microg/kg from noncontaminated (blank) samples. Investigated concentration ranges were 880-3600 microg/kg for ergosterol and 310-2596 microg/kg for DON. The percentage of correctly classified samples was up to 100% for individual samples compared with a number of blank samples.     

Innovative approach to investigating the microstructure of calcified tissues using specular reflectance Fourier transform-infrared microspectroscopy and discriminant analysis

Although bone fracture has become a serious global health issue, current clinical assessments of fracture risk based on bone mineral density are unable to accurately predict whether an individual is likely to suffer a fracture. There is increasing recognition that the chemical structure and composition, or microstructure, of mineralized tissues has an important role to play in determining the fracture resistance of bone. The objective of this preliminary study was to evaluate the use of specular reflectance Fourier transform infrared (SR FT-IR) microspectroscopy in conjunction with discriminant analysis as an innovative technique for providing future insights into the origins of orthopedic abnormalities. The impetus for this approach was that SR FT-IR microspectroscopy would offer several advantages over conventional transmission methods. Bone samples were obtained from young racehorses at known fracture predilection sites and spectra were successfully obtained from calcified cartilage and subchondral bone for the first time. By applying discriminant analysis to the spectral data set in biologically relevant regions, microstructural differences between groups of individuals were found to be related to features associated with both the mineral and organic components of the bone. The preliminary findings also suggest that differences in bone microstructure may exist between healthy individuals of the same age, raising important questions around the normal limits of individual variation and whether individuals may be predisposed to later fracture as a result of detrimental microstructural changes during early growth and development.     

Cosine histogram analysis for spectral image data classification

Conventional multivariate strategies for making qualitative estimates of sample composition rely chiefly on identifying subtle differences in spectral shape. In some instances, such as in biological tissues, the spectra obtained from a single sample class may consist of many shapes. Likewise, two distinctly different sample classes, such as normal and abnormal tissue, may produce similar variations in spectral shape. In our work, we employ statistical analysis of the set of cosine correlation scores obtained from multispectral visible absorption images of stained cervical Papanicolaou samples. By analyzing the cosine correlation score frequency for spectra obtained from the cell nuclei, abnormal cells can be differentiated from the background of normal cells, which vary considerably in their optical properties and morphology. Our method, called cosine histogram analysis (CHA), returns the percent likelihood of abnormality for each pixel in the field of view and is presented here for the first time.     

Regional difference of water content in human skin studied by diffuse-reflectance near-infrared spectroscopy: consideration of measurement depth

Diffuse reflectance (DF) spectra in the 1250-2500 nm region were measured in vivo for the skin of the forehead, cheek, jaw, elbow, volar forearm, palm, knee, and heel of seven healthy volunteers, using a Fourier transform near-infrared (FT-NIR) spectrophotometer with a fiber-optic probe. Apparent regional differences of water content in the skin, as estimated from the diffuse reflectance NIR spectra, are discussed in relation to the influence of measurement depth. The NIR spectra were collected with or without a 300 microm gap between the fiber-optic probe and the skin surface. For comparison, in vitro NIR spectra of stratum corneum sheets equilibrated at 41, 50, 63, and 81% relative humidity, at 25 degrees C, were also obtained. There was a difference in the ratio of the two water bands centered near 1450 nm and 1900 nm between the contact and non-contact measurements. In addition, regional differences of water content calculated from the peak height of the 1900 nm water band, which was normalized to the peak height of the 2175 nm amide band, were compared. The results of Monte Carlo simulation indicated that the apparent regional differences arise at least in part from differences in the measurement depth due to differences in specular reflection at the skin surface and in the thickness of the stratum corneum.     

The Use of Gas-Phase UV Spectra in the 168-330-nm Wavelength Region for Analytical Purposes. 1. Qualitative Measurements

A novel instrument, which combines gas chromatographic separation with ultraviolet detection, has been employed in the qualitative study of molecular UV absorption spectra in the gas phase. The wavelength range available with the current instrument made studies in the region of 168-330 nm possible. The absorption spectra between 168 and 330 nm for ～1000 organic compounds and a number of inorganic gases were registered. For all the spectra recorded, 69% showed absorption maxima shorter than 190 nm, and as much as 87% shorter than 200 nm. This indicates the importance of the short UV wavelength region for analytical purposes. The results showed that each compound studied gave rise to unique absorption spectra. The influence of temperature on the shape of the spectra was studied in the range of 15-205 °C. A slight broadening effect on spectral absorption bands (0.3 nm) and the vibrational structure (maximally 1.4 nm) with increased temperature was observed. However, this effect was shown to have no influence on the analytical results. In the vast majority of cases, where structural isomers were investigated, there were clear differences between the recorded spectra. Compounds with the same functional group showed pronounced similarities. The influence of conjugation and various substituents could be demonstrated. A commercial computer program was used for a computer search of unknowns against the recorded reference spectra. This showed that the gas-phase UV spectra are very well defined and that a search always resulted in a very close agreement, even if concentrations and chromatographic conditions were different between the recording of reference and unknown. When a search was performed on a compound not present in the library, the result pointed out the correct class of compounds.     

Variations in predicted values from near-infrared spectra of samples in vials by using a calibration model developed from spectra of samples in vials: causes of the variations and compensation methods

The causes and compensation methods of variations in the predicted values from near-infrared (NIR) spectra of samples in vials by using a calibration model developed from spectra of samples in vials were studied. From the viewpoint of the path length and the peak height of spectra in the NIR region, vials with 1 mL volume (external diameter: 8 mm, inside diameter: about 6 mm, length: 40 mm) were selected as disposable vials for the samples. To investigate the causes of the variations in the predicted values, the optical path lengths and transmittance spectra of empty vials in five lots produced during the last four years were examined. The results showed that there were some differences in the path lengths of the vials and in the intensities of peaks near 7130 and 4515 cm-1, which were attributed to the hydroxyl groups in the spectra of the vials among the five lots. The calibration models for the concentration of isopropyl alcohol (IPA) in toluene contained in vials of the same lot were developed. To search the pretreatment methods for the best calibration model with small variations in the predictive values of the samples in the vials, six kinds of pretreatments (no pretreatment, baseline correction, first derivative, second derivative, multiplicative scattering correction (MSC), and standard normal variate (SNV)) were compared. The results indicated that MSC is a very effective method with small variation in the predicted values from the spectra of samples in vials. It was found that the variations in the predicted values are caused mainly by variations in the path lengths of vials. In real quality control, the NIR-chemometrics method is requested for long-term traceability and good reproducibility of the predictive value. Therefore, it is crucial to watch changes in the path length under a fixed path length as a reference. From this standpoint, modified MSC, which uses a particular spectrum as a reference instead of the average spectrum of a sample set, was proposed in the present study, and it was demonstrated that the variations due to the individual differences in the vials can not only be compensated, but the path lengths of vials could also be estimated by the modified MSC. Moreover, a calibration model for the toluene concentration in silicone oil was developed to confirm the effects of modified MSC by using another sample set. The results showed that the compensation of vial path lengths using the modified MSC is also useful for other samples, similar to the case of the sample of IPA in toluene.