Blue-violet excited autofluorescence spectroscopy and imaging of normal and cancerous human bronchial tissue after formalin fixation

Autofluorescence (AF) imaging is a powerful tool for the detection of (pre-)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre-)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin-fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre-)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre-)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studies.     

A normal mode-based geometric simulation approach for exploring biologically relevant conformational transitions in proteins

A three-step approach for multiscale modeling of protein conformational changes is presented that incorporates information about preferred directions of protein motions into a geometric simulation algorithm. The first two steps are based on a rigid cluster normal-mode analysis (RCNMA). Low-frequency normal modes are used in the third step (NMSim) to extend the recently introduced idea of constrained geometric simulations of diffusive motions in proteins by biasing backbone motions of the protein, whereas side-chain motions are biased toward favorable rotamer states. The generated structures are iteratively corrected regarding steric clashes and stereochemical constraint violations. The approach allows performing three simulation types: unbiased exploration of conformational space; pathway generation by a targeted simulation; and radius of gyration-guided simulation. When applied to a data set of proteins with experimentally observed conformational changes, conformational variabilities are reproduced very well for 4 out of 5 proteins that show domain motions, with correlation coefficients r > 0.70 and as high as r = 0.92 in the case of adenylate kinase. In 7 out of 8 cases, NMSim simulations starting from unbound structures are able to sample conformations that are similar (root-mean-square deviation = 1.0-3.1 ?) to ligand bound conformations. An NMSim generated pathway of conformational change of adenylate kinase correctly describes the sequence of domain closing. The NMSim approach is a computationally efficient alternative to molecular dynamics simulations for conformational sampling of proteins. The generated conformations and pathways of conformational transitions can serve as input to docking approaches or as starting points for more sophisticated sampling techniques.     

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.     

ICE: a new method for the multivariate curve resolution of hyperspectral images

The iterated constrained endmembers (ICE) algorithm is a new method of unmixing hyperspectral images that combines aspects of multivariate curve resolution (MCR) methods in chemometrics and unmixing algorithms in remote sensing. Like many MCR methods, ICE also estimates pure components, or endmembers, via alternating least squares; however, it is explicitly based on a convex geometry model and estimation is carried out in a subspace of reduced dimensionality defined by the minimum noise fraction (MNF) transform. In this paper, we describe the ICE algorithm and its properties. We also illustrate its use on a hyperspectral image of cervical tissue. The unmixing of hyperspectral images presents some unique challenges, and we also outline where further development is required. © 2008 John Wiley & Sons, Ltd.     

Tailoring approach for exploring electron densities and electrostatic potentials of molecular crystals

Experimental and theoretical studies of electron densities and the corresponding derived entities such as electrostatic potentials have been the primary means of understanding the chemical nature and electronic properties of crystalline substances. Conventional crystal calculation methods such as the embedded cluster models are capable of performing calculations on small and medium-sized molecules, while periodic ab initio methods can treat crystals with up to 200 atoms per unit cell. A linear scaling method, viz. the molecular tailoring approach, has recently been developed for obtaining ab initio quality one-electron properties. In the present study, the molecular tailoring approach is employed to generate electron density, electrostatic potential and interaction density maps with the ibuprofen crystal as a test case. The interaction density and electrostatic potential maps produced in the present work succinctly bring out the actual crystalline environment around a given reference molecule by including the interactions with atoms in its neighborhood. The results obtained from the molecular tailoring approach may thus be expected to enhance our understanding of the environment in the crystalline material with reasonably small computational effort.Contribution to the Jacopo Tomasi Honorary Issue     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Nonphotochemical hole-burning study of selectively stained normal and cancerous human ovarian tissues

Results are presented of nonphotochemical hole-burning (HB) experiments on cancerous ovarian and analogous normal peritoneal in vitro tissues stained with the mitochondrial-selective dye rhodamine 800. A comparison of fluorescence excitation spectra, hole-growth kinetics data, and external electric field (Stark) effects on the shape of spectral holes burned in cancerous and normal tissues stained with rhodamine 800 revealed significant differences only in the dipole moment change (fDeltamu) measured by a combination of HB and Stark spectroscopies. It is shown that the permanent dipole moment change for the S0--> S1 transition of the rhodamine 800 molecules in cancerous tissue is higher than that of normal tissue by a factor of about 1.4. The finding is similar to the HB results obtained earlier for human ovarian surface epithelial cell lines, i.e., OV167 carcinoma and OSE(tsT)-14 normal cells stained with the same mitochondria-specific dye (Walsh et al. Biophys. J. 2003, 84, 1299). We propose that the observed difference in the permanent dipole moment change in cancerous ovarian tissue is related to a modification in mitochondrial membrane potential.     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Estimation of trace vapor concentration-pathlength in plumes for remote sensing applications from hyperspectral images

A novel approach for quantification of chemical vapor effluents in stack plumes using infrared hyperspectral imaging are presented and examined. The algorithms use a novel application of the extended mixture model to provide estimates of background clutter in the on-plume pixel. These estimates are then used iteratively to improve the quantification. The final step in the algorithm employs either an extended least-squares (ELS) or generalized least-squares (GLS) procedure. It was found that the GLS weighting procedure generally performed better than ELS, but they performed similarly when the analyte spectra had relatively narrow features. The algorithms require estimates of the atmospheric radiance and transmission from the target plume to the imaging spectrometer and an estimate of the plume temperature. However, estimates of the background temperature and emissivity are not required which is a distinct advantage. The algorithm effectively provides a local estimate of the clutter, and an error analysis shows that it can provide superior quantification over approaches that model the background clutter in a more global sense. It was also found that the estimation error depended strongly on the net analyte signal for each analyte, and this quantity is scenario-specific.     

Instrument design and characterization for high resolution MALDI-MS imaging of tissue sections

In previous work, we have reported using a MALDI imaging time-of-flight mass spectrometer for the detection of protein ions from tissue sections with spatial resolution of 25 microm. We present here imaging mass spectrometry results obtained with a high-resolution scanning MALDI time-of-flight mass spectrometer, equipped with a coaxial laser illumination ion source, capable of achieving irradiation areas as small as 40 microm(2) (ca 7 microm diameter). MALDI-generated analyte ion signals from these very small irradiation volumes can be observed in a molecular weight range up to 27,000. High-resolution imaging mass spectrometry images were successfully generated from matrix thin film samples and tissue sections with scanning resolutions at and below 10 microm. This work also provides fundamental characterization of the ion signal dependence as a function of various focus and fluence parameters that will be required for extension to tissue imaging at the subcellular level.     

Resolution and segmentation of hyperspectral biomedical images by multivariate curve resolution-alternating least squares

MCR-ALS is a resolution method that has been applied in many different fields, such as process analysis, environmental data and, recently, hyperspectral image analysis. In this context, the algorithm provides the distribution maps and the pure spectra of the image constituents from the sole information in the raw image measurement. Based on the distribution maps and spectra obtained, additional information can be easily derived, such as identification of constituents when libraries are available or quantitation within the image, expressed as constituent signal contribution. This work summarizes first the protocol followed for the resolution on two examples of kidney calculi, taken as representations of images with major and minor compounds, respectively. Image segmentation allows separating regions of images according to their pixel similarity and is also relevant in the biomedical field to differentiate healthy from non-healthy regions in tissues or to identify sample regions with distinct properties. Information on pixel similarity is enclosed not only in pixel spectra, but also in other smaller pixel representations, such as PCA scores. In this paper, we propose the use of MCR scores (concentration profiles) for segmentation purposes. K-means results obtained from different pixel representations of the data set are compared. The main advantages of the use of MCR scores are the interpretability of the class centroids and the compound-wise selection and preprocessing of the input information in the segmentation scheme.     

Efficient encoding and rapid decoding for interactive visualization of large three‐dimensional hyperspectral chemical images

Interactive visualization of data from a new generation of chemical imaging systems requires coding that is efficient and accessible. New technologies for secondary ion mass spectrometry (SIMS) generate large three‐dimensional, hyperspectral datasets with high spatial and spectral resolution. Interactive visualization is important for chemical analysis, but the raw dataset size exceeds the memory capacities of typical current computer systems and is a significant obstacle. This paper reports the development of a lossless coding method that is memory efficient, enabling large SIMS datasets to be held in fast memory, and supports quick access for interactive visualization. The approach provides pixel indexing, as required for chemical imaging applications, and is based on the statistical characteristics of the data. The method uses differential time‐of‐flight to effect mass‐spectral run‐length‐encoding and uses a scheme for variable‐length, byte‐unit representations for both mass‐spectral time‐of‐flight and intensity values. Experiments demonstrate high compression rates and fast access. Copyright © 2009 John Wiley & Sons, Ltd.     

A Comparison of DESI-MS and LC-MS for the Lipidomic Profiling of Human Cancer Tissue

In this study, we make a direct comparison between desorption electrospray ionization-mass spectrometry (DESI-MS) and ultraperformance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) platforms for the profiling of glycerophospholipid (GPL) species in esophageal cancer tissue. In particular, we studied the similarities and differences in the range of GPLs detected and the congruency of their relative abundances as detected by each analytical platform. The main differences between mass spectra of the two modalities were found to be associated with the variance in adduct formation of common GPLs, rather than the presence of different GPL species. Phosphatidylcholines as formate adducts in UPLC-ESI-MS accounted for the majority of differences in negative ion mode and alkali metal adducts of phosphatidylcholines in DESI-MS for positive ion mode. Comparison of the relative abundance of GPLs, normalized to a common peak, revealed a correlation coefficient of 0.70 (P < 0.001). The GPL profile detected by DESI-MS is congruent to UPLC-ESI-MS, which reaffirms the role of DESI-MS for lipidomic profiling and a potential premise for quantification.

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Graphical Abstract ?

     

Multielemental determination in normal,benign, and cancerous tissues of the human brain

Although the metal content of the human body is only about 3%, metals are very important for human lives. Diseases occur when an excess or deficiency of in-vivo metals appear, when other metal pollutants enter the body, or when poisons or viruses enter into the metal ligand competition. Cancer is caused by carcinogens, which are substances capable of producing tumors in any test species at any dose level. This paper discusses the determination of some elements in diseased tissues of the human brain. As the elements present are mostly at micro- or nano-gram levels, the very sensitive technique of neutron activation analysis involving radiochemical separation has been employed. Substocihiometric estimations were carried out wherever possible. The radiochemical separation procedure includes a solvent extraction and precipitation technique. The elements estimated in the tissue samples are Cu, Au, As, Se, Hg, Co, Zn, Ca, Fe, P, Cr, Na, and K. The accuracy, precision, and radiochemical purity of the method have been discussed. Two samples and a standard can be analyzed in four days.     

GC–MS method for the determination of paraben preservatives in the human breast cancerous tissue

The general presumption that the preservative laden personal care products may be one of the causative agents for breast cancer, has remained a matter of controversy during this decade. Extensive studies have not been carried out to either prove or disprove the role of preservatives in breast cancer incidences. In this study we have developed a new method for the identification and quantification of the preservatives such as methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP) in breast tissue using Gas Chromatography and Mass Spectrometry (GC–MS). Tissue was extracted by using acetone:n-hexane mixture (1:1 v/v) and derivatized with N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The extent of reaction time and the amount of MSTFA to attain greater derivatization were optimized. The developed method yielded good recovery (mean ± SD) of 99.8 ± 5.1, 96 ± 4.4, 107 ± 17 and 113 ± 13% with relative standard deviations (RSDs) of 5.1, 4.6, 15.6 and 13%, and the limits of detection (LOD) of 2.02, 1.05, 1.71 and 3.75 ng g^{− 1} for MeP, EtP, PrP and BuP, respectively. The method was successfully validated for the determination of parabens including butyl paraben (log K_ow = 3.57) in cancerous breast tissues; this could be a promising one for screening of breast tissues and also the environment for paraben residues. As far as our knowledge goes this is the first GC–MS method for the determination of parabens in human tissue.     

Interactive effects of ultraviolet-B radiation and temperature on cotton physiology, growth, development and hyperspectral reflectance

Current conditions of 2-11 kJ m(-2) day(-1) of UV-B radiation and temperatures of >30 degrees C during flowering in cotton cultivated regions are projected to increase in the future. A controlled environment study was conducted in sunlit growth chambers to determine the effects of UV-B radiation and temperature on physiology, growth, development and leaf hyperspectral reflectance of cotton. Plants were grown in the growth chambers at three day/night temperatures (24/16 degrees C, 30/22 degrees C and 36/28 degrees C) and three levels of UV-B radiation (0, 7 and 14 kJ m(-2) day(-1)) at each temperature from emergence to 79 days under optimum nutrient and water conditions. Increases in main stem node number and the node of first fruiting branch and decrease in duration to first flower bud (square) and flower were recorded with increase in temperature. Main effects of temperature and UV-B radiation were significant for net photosynthetic rates, stomatal conductance, total chlorophyll and carotenoid concentrations of uppermost, fully expanded leaves during squaring and flowering. A significant interaction between temperature and UV-B radiation was detected for total biomass and its components. The UV-B radiation of 7 kJ m(-2) day(-1) reduced boll yield by 68% and 97% at 30/22 degrees C and 36/28 degrees C, respectively, compared with yield at 0 kJ m(-2) day(-1) and 30/22 degrees C. No bolls were produced in the three temperature treatments under 14 kJ m(-2) day(-1) UV-B radiation. The first-order interactions between temperature, UV-B radiation and leaf age were significant for leaf reflectance. This study suggests a growth- and process-related temperature dependence of sensitivity to UV-B radiation.     

Biomechanical analysis of cancerous and normal cells based on bulge generation in a microfluidic device

This paper presents a new biomechanical analysis method for discrimination between cancerous and normal cells through compression by poly(dimethylsiloxane) (PDMS) membrane deflection in a microfluidic device. When a cell is compressed, cellular membrane will expand and then small bulges will appear on the peripheral cell membrane beyond the allowable strain. It is well known that the amount of F-actin in cancer cells is less than that of normal cells and bulges occur at the sites where cytoskeleton becomes detached from the membrane bilayer. Accordingly, we have demonstrated the difference of the bulge generation between breast cancer cells (MCF7) and normal cells (MCF10A). After excessive deformation, the bulges generated in MCF7 cells were not evenly distributed on the cell periphery. Contrary to this, the bulges of MCF10A cells showed an even distribution. In addition, the morphologies of bulges of MCF7 and MCF10A cells looked swollen protrusion and tubular protrusion, respectively. Peripheral strains at the moment of the bulge generation were also 72% in MCF7 and 46% in MCF10A. The results show that the bulge generation can be correlated with the cytoskeleton quantity inside the cell, providing the first step of a new biomechanical approach.     

Densitometry of photomicrographic negatives for the determination of fluorophores in sections of tissue

Concentrations of fluorophores in histological sections can be determined by measuring the optical density of photomicrographic negatives of the fluorescence emission. This method allows measurements to be made with high resolution and high sensitivity. It is necessary to determine the relationship between fluorophore concentration and emission intensity, the effects of spatial and temporal variations in the intensity of the exciting illumination, and the characteristic curve of each film. A study of these problems with two model systems is described and the procedures necessary to obtain accurate and precise results are reviewed.     

1H NMR-based metabolomics approach for exploring urinary metabolome modifications after acute and chronic physical exercise

Metabolomics is a comprehensive method for metabolite assessment that involves measuring the overall metabolic signature of biological samples. We used this approach to investigate biochemical changes due to acute and chronic physical exercise. Twenty-two women using identical oral contraceptives were segregated into an untrained (n = 10) or trained (n = 12) group depending on their physical training background. The subjects performed two exercises in a randomized order: a prolonged exercise test (75% of their \mathop V^· \textO_2   max \mathop V\limits^\cdot {{\text{O}}_{2\,\;\max }} until exhaustion) and a short-term, intensive exercise test (short-term, intensive exercise anaerobic test). Urine specimens were collected before and 30 min after each test. The samples were analyzed by ¹H NMR spectroscopy, and multivariate statistical techniques were utilized to process the data. Distinguishing characteristics were observed only in the urine profiles of specimens collected before vs. 30 min after the short-term, intensive exercise test. The metabolites responsible for such changes were creatinine, lactate, pyruvate, alanine, β-hydroxybutyrate, acetate, and hypoxanthine. In both groups, the excretion of lactate, pyruvate, alanine, β-hydroxybutyrate, and hypoxanthine increased similarly after the completion of the short-term, intensive exercise test (p < 0.03). However, acetate excretion increased to a lesser extent in trained than in untrained subjects (p < 0.05). In conclusion, metabolomics is a promising tool in order to gain insight into physiological status and to clarify the changes induced by short-term, intense physical exercise.