Multivariate statistical identification of human bladder carcinomas using ambient ionization imaging mass spectrometry

Diagnosis of human bladder cancer in untreated tissue sections is achieved by using imaging data from desorption electrospray ionization mass spectrometry (DESI-MS) combined with multivariate statistical analysis. We use the distinctive DESI-MS glycerophospholipid (GP) mass spectral profiles to visually characterize and formally classify twenty pairs (40 tissue samples) of human cancerous and adjacent normal bladder tissue samples. The individual ion images derived from the acquired profiles correlate with standard histological hematoxylin and eosin (H&E)-stained serial sections. The profiles allow us to classify the disease status of the tissue samples with high accuracy as judged by reference histological data. To achieve this, the data from the twenty pairs were divided into a training set and a validation set. Spectra from the tumor and normal regions of each of the tissue sections in the training set were used for orthogonal projection to latent structures (O-PLS) treated partial least-square discriminate analysis (PLS-DA). This predictive model was then validated by using the validation set and showed a 5% error rate for classification and a misclassification rate of 12%. It was also used to create synthetic images of the tissue sections showing pixel-by-pixel disease classification of the tissue and these data agreed well with the independent classification that uses histological data by a certified pathologist. This represents the first application of multivariate statistical methods for classification by ambient ionization although these methods have been applied previously to other MS imaging methods. The results are encouraging in terms of the development of a method that could be utilized in a clinical setting through visualization and diagnosis of intact tissue.     

Crisp and soft multivariate methods visualize individual cell nuclei in Raman images of liver tissue sections

Raman and infrared spectroscopy have been recognized to be promising tools in clinical diagnostics because they provide molecular contrast without external stains. Here, vertex component analysis (VCA) was applied to Raman and Fourier transform infrared (FTIR) images of liver tissue sections and the results were compared with K-means cluster analysis, fuzzy C-means cluster analysis and principal component analysis. The main components of VCA from three Raman images were assigned to the central vein, periportal vein, cell nuclei, liver parenchyma and bile duct. After resonant Mie scattering correction, VCA of FTIR images identified veins, liver parenchyma, cracks, but no cell nuclei. The advantages of VCA in the context of tissue characterization by vibrational spectroscopic imaging are that the tissue architecture is visualized and the spectral information is reconstructed. Composite images were constructed that revealed a high molecular contrast and that can be interpreted in a similar way like hematoxylin and eosin stained tissue sections.     

Characterization of low molecular weight hydrocarbon oligomers by laser desorption/ionization time-of-flight mass spectrometry using a solvent-free sample preparation method.

A new solvent-free sample preparation method using silver trifluoroacetate (AgTFA) was developed for the analysis of low molecular weight paraffins and microcrystalline waxes by laser desorption/ionization time-of-flight mass spectrometry (LDI-TOFMS). Experiments show that spectral quality can be enhanced by dispersing AgTFA directly in liquid paraffins without the use of additional solvents. This preparation mixture is applied directly to the MALDI probe. Solid waxes could be examined by melting prior to analysis. The method also provides sufficiently reproducible spectra that peak area ratios between mono- and bicyclic alkane peaks indicated variations in the cycloalkane content of paraffin samples. Dehydrogenation of hydrocarbons observed during the desorption/ionization process was studied by analysis of alkane standards.     

Preparation of tissue samples for X-ray fluorescence microscopy

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level.

The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined.

Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg.

The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.     

A new method of comparing 2D-PAGE maps based on the computation of Zernike moments and multivariate statistical tools

The aim of this work was to obtain the correct classification of a set of two-dimensional polyacrylamide gel electrophoresis map images using the Zernike moments as discriminant variables. For each 2D-PAGE image, the Zernike moments were computed up to a maximum p order of 100. Partial least squares discriminant analysis with variable selection, based on a backward elimination algorithm, was applied to the moments calculated in order to select those that provided the lowest error in cross-validation. The new method was tested on four datasets: (1) samples belonging to neuroblastoma; (2) samples of human lymphoma; (3) samples from pancreatic cancer cells (two cell lines of control and drug-treated cancer cells); (4) samples from colon cancer cells (total lysates and nuclei treated or untreated with a histone deacetylase inhibitor). The results demonstrate that the Zernike moments can be successfully applied for fast classification purposes. The final aim is to build models that can be used to achieve rapid diagnosis of these illnesses.     

Terahertz time-domain spectroscopy of some pentoses

1 Introduction Terahertz time-domain spectroscopy (THz-TDS) is a new spectral technique based on femto-second laser technology developed since the 1980s[1]. Terahertz (THz) waves lie between the infrared and microwave bands in the electromagnetic spectrum covering the frequency range from 100 GHz to 10 THz. Despite great scientific interest, the THz frequency range re- mains one of the least tapped regions of the electro- magnetic spectrum because there are neither conven- ient high-power…     

Sublimation as a method of matrix application for mass spectrometric imaging

Common organic matrix-assisted laser desorption/ionization (MALDI) matrices, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and alpha-cyano-4-hydroxycinnamic acid, were found to undergo sublimation without decomposition under conditions of reduced pressure and elevated temperature. This solid to vapor-phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temperature, and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid standards robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcrystalline morphology of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible and eliminated the potential for spreading of analytes arising from solvent deposition during matrix application.     

Monte Carlo simulation of near infrared autofluorescence measurements of in vivo skin

The autofluorescence properties of normal human skin in the near-infrared (NIR) spectral range were studied using Monte Carlo simulation. The light-tissue interactions including scattering, absorption and anisotropy propagation of the regenerated autofluorescence photons in the skin tissue were taken into account in the theoretical modeling. Skin was represented as a turbid seven-layered medium. To facilitate the simulation, ex vivo NIR autofluorescence spectra and images from different skin layers were measured from frozen skin vertical sections to define the intrinsic fluorescence properties. Monte Carlo simulation was then used to study how the intrinsic fluorescence spectra were distorted by the tissue reabsorption and scattering during in vivo measurements. We found that the reconstructed model skin spectra were in good agreement with the measured in vivo skin spectra from the same anatomical site as the ex vivo tissue sections, demonstrating the usefulness of this modeling. We also found that difference exists over the melanin fluorescent wavelength range (880-910 nm) between the simulated spectrum and the measured in vivo skin spectrum from a different anatomical site. This difference suggests that melanin contents may affect in vivo skin autofluorescence properties, which deserves further investigation.     

Optical diagnosis of peritoneal metastases by infrared microscopic imaging

Fourier transform infrared (FTIR) spectroscopy is nowadays widely accepted as a technique with high potential for diagnosis of cancerous tissues. This study presents an example of the investigation of peritoneal metastases by FTIR microimaging. Peritoneal malignancies are generally secondary localizations of primary visceral cancers such as ovarian, stomach or colon cancers. By analysing simultaneously both formalin-fixed paraffin-embedded and frozen specimens, we examined malignant and non-malignant (i.e. fibrotic and cicatricial) peritoneal lesions. Paraffin-embedded tissues were analysed without any previous dewaxing. Multivariate statistical approaches, based on the classification of infrared data by hierarchical cluster analysis, allowed the discrimination of these various samples. Microimaging also permits the revelation of the heterogeneity of the tissue: it was possible to localize precisely the cancerous areas, and to distinguish, on the basis of their spectral signatures, the peritumoral neighbouring connective tissue close to the carcinomatous areas from the connective tissue distant from the cancerous areas. These spectral differences could be useful as complementary information to study molecular changes associated with the malignancy.     

涂石蜡大米傅立叶变换红外吸收光谱识别分析

针对11种未涂石蜡大米和18个涂不同量石蜡的大米样品,以石油醚提取的油脂为试样,采用傅立叶变换红外光谱仪,扫描样品的傅立叶变换红外吸收光谱,并对光谱进行预处理,提取红外特征信息,将2855与1746、1462与1163 cm-1处特征峰的面积比值为坐标,采用Origín 6.0软件作识别分类图.结果表明:特征峰的面积比值与所涂石蜡量成线性变化,大米油脂的特征峰面积比值在一定的区域分布,涂以0.05%以上石蜡的大米,其油脂特征峰面积比值与未涂石蜡米油脂的值有一定区别.     

Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.     

基于人工神经网络的生物组织质谱成像分类与识别方法

生物组织质谱成像技术不仅能够展示组织的生物分子信息,而且能直观地显示分子空间分布,是当今生物质谱的研究热点.如何对生物组织质谱成像的数据进行基于生物分子的有效分类与识别是该领域关注的重要问题,特别对于病变组织与其邻近非病变组织的区分与识别和生物组织功能区域的划分与鉴定具有重要的意义.本研究开发出一种新的分类与识别方法.其流程是,首先进行质谱成像数据预处理,应用无监督的自组织特征映射网络区分组织样品区与非组织区域,提取组织区域的质谱数据,应用有监督的学习向量量化网络对已知类别数据进行学习训练,建立模型;应用模型对未知样品进行识别.应用本方法对6个膀胱癌患者的膀胱癌变组织与邻近非癌变组织的质谱成像数据进行分类与识别,结果显示,癌变组织判错率低于23.38％,而非癌变组织判错率低于9.08％,表现出较高的准确度;对3片邻近的小鼠大脑切片质谱成像数据进行白质与灰质区域划分,将中间的1片用于训练,两边的2片用于验证,结果显示,自组织特征映射网络的分类结果与学习向量量化网络的预测结果不一致率低于4％.本方法基于生物分子的质谱成像组织区域分类与识别,具有较高准确度和操作简便等优点,在临床医学研究领域有大规模的应用潜能.     

Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging

Infrared (IR) spectroscopic imaging coupled with microscopy has been used to investigate thin sections of cervix uteri encompassing normal tissue, precancerous structures, and squamous cell carcinoma. Methods for unsupervised distinction of tissue types based on IR spectroscopy were developed. One-hundred and twenty-two images of cervical tissue were recorded by an FTIR spectrometer with a 64×64 focal plane array detector. The 499,712 IR spectra obtained were grouped by an approach which used fuzzy C-means clustering followed by hierarchical cluster analysis. The resulting false color maps were correlated with the morphological characteristics of an adjacent section of hematoxylin and eosin-stained tissue. In the first step, cervical stroma, epithelium, inflammation, blood vessels, and mucus could be distinguished in IR images by analysis of the spectral fingerprint region (950–1480 cm⁻¹). In the second step, analysis in the spectral window 1420–1480 cm⁻¹ enables, for the first time, IR spectroscopic distinction between the basal layer, dysplastic lesions and squamous cell carcinoma within a particular sample. The joint application of IR microspectroscopic imaging and multivariate spectral processing combines diffraction-limited lateral optical resolution on the single cell level with highly specific and sensitive spectral classification on the molecular level. Compared with previous reports our approach constitutes a significant progress in the development of optical molecular spectroscopic techniques toward an additional diagnostic tool for the early histopathological characterization of cervical cancer.     

Perspectives in tissue microarrays

Tissue microarrays (TMAs) are means of combining tens to hundreds of specimens of tissue onto a single slide for analysis at one time. TMAs are most frequently constructed from paraffin embedded tissue; however, they can be constructed from frozen tissue. The construction of TMAs is flexible, meeting the focused needs of the investigator. A TMA slide can be processed like an ordinary tissue section, and used for histochemical, immunohistochemical staining or in situ hybridization. Combined with automated new image analysis systems, TMAs are a powerful molecular profiling tool. By confirming the findings of microarray experiments or protein arrays, TMAs can be applied systematically to global cellular network analysis within tissue cell. TMAs are commonly used to confirm the results of expression microarrays as well as in the development of diagnostic and prognostics markers for clinical applications. This review will cover recent advancements in technology for the construction and use of TMAs. Because TMAs can be constructed from archival paraffin embedded tissue, they open up the vast archive of patient samples and make them accessible for medical research. TMAs play an ever increasing role in translational medicine, bridging the chasm of discovery at the research bench to the demonstration of clinical utility prior to implementation in patient care.     

Determination of pharmaceutical compounds in skin by imaging matrix-assisted laser desorption/ionisation mass spectrometry

Matrix-assisted laser desorption/ionisation (MALDI) quadrupole time-of-flight mass spectrometry (Q-TOFMS) has been used to detect and image the distribution of a xenobiotic substance in skin. Porcine epidermal tissue was treated with 'Nizoral', a medicated shampoo containing ketoconazole (+/-)-1-acetyl-4-[p-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) as active ingredient. Following incubation for 1 h at 37 degrees C all excess formulation was washed from the surface. A cross-section of the drug-treated tissue was then blotted onto a cellulose membrane, precoated in matrix (alpha-cyano-4-hydroxycinnamic acid (CHCA)), by airspray deposition. In separate experiments the tissue surface was treated with Nizoral within a triangular former, and subsequently blotted onto a matrix-coated membrane. Sample membranes were then mounted into the recess of specialised MALDI targets with adhesive tape. All samples were analysed by MALDI-TOFMS using an Applied Biosystem 'Q-star Pulsar i' hybrid Q-TOF mass spectrometer fitted with an orthagonal MALDI ion source and imaging software. Detection of the protonated molecule was readily achievable by this technique. Treatment of the tissue within a template gave rise to images depicting the expected distribution of the drug, demonstrating that this technique is capable of producing spatially useful data. Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross-sectional imprint of treated tissue. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Superimposing the mass spectrometric and histological images appeared to indicate drug permeation into the dermal tissue layer.     

Distribution of metal-free sulfonated phthalocyanine in subcutaneously transplanted murine tumors

Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H(2)PcS(2.4)) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H(2)PcS(2.4) with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H(2)PcS(2.4) intravenous injection revealed that H(2)PcS(2.4) was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H(2)PcS(2.4) was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H(2)PcS(2.4) less than adjacent skin structures, whereas EC and LLC accumulated H(2)PcS(2.4) alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H(2)PcS(2.4) accumulation. The H(2)PcS(2.4) concentration in remote healthy tissues (skin, muscles and connective tissue) was 2-3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H(2)PcS(2.4) predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.     

Study of normal colorectal tissue by FT-Raman spectroscopy

FT-Raman spectroscopy was employed to study normal human colorectal tissues in vitro with the aim of evaluating the spectral differences of the complex colon mucous in order to establish a characteristic Raman spectrum. The samples were collected from 39 patients, providing 144 spectra for the statistical analysis. The results enable one to estabilish three well-defined spectroscopic groups of non-altered coloretal tissues that were consistently checked by statistical (clustering) and biological (histopathology) analyses: group 1 is represented by samples with the presence of epithelial layer, connective tissue papillae, and smooth muscle tissue; group 2 comprises tissues with epithelial layer and connective tissue papillae; group 3 presented mostly fatty and slack conjunctive tissue. The study reveals the existence of an intrinsic spectral variability for each patient that must be considered when sampling tissues fragments to build a spectral database. This is the first step for future studies and applications of Raman spectroscopy to optical biopsy and diagnosis of colorectal cancer.     

Elemental composition of skin tissue by PIXE and INA analyses

Hypertrophic scars are a particular type of scar that can form after any type of dermal injury. They are unsightly, red, elevated above nomal skin level, itchy and painful. At present no-one knows why these scars form. Due to the differences between scar tissue and normal skin it was decided to analyse skin tissue which at a later date would be compared to scar tissue. Split skin graft (epidemis) samples as well as full thickness skin (epidemis to demis) tissue were analysed using PIXE and RBS with a 2 MeV proton beam. The epidemis was compared to the demis to see if there were any variations between the two. In all the samples P, S, Cl, K, Ca, Fe, and Cd were detected and in a majority of the samples Zn and Ba were found, using PIXE. It was found that, between the epidemis and demis, there was an significant increase in P in the epidermis in both full thickness and split skin graft samples. Complementary elemental analysis was also performed using neutron activation, and Br, Na and Zn were detected.     

红外光谱分析数据特征指纹的可视化表达方法

提出一类红外光谱分析数据特征指纹的可视化表达方法。该方法采用基于贝叶斯准则的多元统计方法对原始数据进行投影变换，再以地维灰度图对变换后数据进行可视化表征，形成基于计算机图像的虚拟化学指纹图谱。将其用于鉴别3种不同产地中药当归样品的结果表明，它能有效提取红外光谱分析数据的特征指纹，实现以虚拟指纹图谱对药材产地的分类鉴别，从而为辨识复杂化学物质体系提供了新的技术手段。