Beny Cohen

A comparative study of gallstones from children and adults using FTIR spectroscopy and fluorescence microscopy

BackgroundCholelithiasis is the gallstone disease (GSD) where stones are formed in the gallbladder. The main function of the gallbladder is to concentrate bile by the absorption of water and sodium. GSD has high prevalence among elderly adults. There are three major types of gallstones found in patients, White, Black and Brown. The major chemical component of white stones is cholesterol. Black and brown stones contain different proportions of cholesterol and bilirubin. The pathogenesis of gallstones is not clearly understood. Analysis of the chemical composition of gallstones using various spectroscopic techniques offers clues to the pathogenesis of gallstones. Recent years has seen an increasing trend in the number of cases involving children. The focus of this study is on the analysis of the chemical composition of gallstones from child and adult patients using spectroscopic methods.MethodsIn this report, we present FTIR spectroscopic studies and fluorescence microscopic analysis of gallstones obtained from 67 adult and 21 child patients. The gallstones were removed during surgical operations at Soroka University Medical Center.ResultsOur results show that black stones from adults and children are rich in bilirubin. Brown stones are composed of varying amounts of bilirubin and cholesterol. Green stones removed from an adult, which is rare, was found to be composed mainly of cholesterol. Our results also indicated that cholesterol and bilirubin could be the risk factors for gallstone formation in adults and children respectively. Fluorescence micrographs showed that the Ca-bilirubinate was present in all stones in different quantities and however, Cu-bilirubinate was present only in the mixed and black stones.ConclusionsAnalysis based on FTIR suggest that the composition of black and brown stones from both children and adults are similar. Various layers of the brown stone from adults differ by having varying quantities of cholesterol and calcium carbonate. Ring patterns observed mainly in the green stone using fluorescence microscopy have relevance to the mechanism of the stone formation. Our preliminary study suggests that bilirubin and cholesterol are the main risk factors of gallstone disease.

Application of FTIR microscopy for the characterization of malignancy: H-ras transfected murine fibroblasts as an example

Recently, microscopic FTIR is widely used in the field of biology and medicine. FTIR can detect biomolecular changes in the cells and tissues responsible for various disorders. In this report, we characterize the H-ras transfected fibroblasts and its normal control using microscopic FTIR. The intensity of the normal fibroblasts was higher than that of H-ras transfected fibroblasts. Our studies showed significant differences occur in the concentration of vital metabolites upon transformation. The DNA and carbohydrates level decreased in the transformed cells compared to the controls. A linear correlation could be found between the levels of carbohydrates and phosphate, while the RNA/DNA ratio varied inversely with glucose/phosphate levels.

Fluorescence spectroscopy for detection of malignancy: H-ras overexpressing fibroblasts as a model.

Autofluorescence from intracellular chromophores upon illumination of cells by monochromatic light has been studied towards the development of novel noninvasive and sensitive technology for the early detection of cancer. To investigate the relationship between biochemical and morphological changes underlying malignant disease and resulting fluorescence spectra, an in vitro model system of a paired normal and malignant murine fibroblasts cell lines, differing in cancer-associated H-ras expression was employed. A comparison of fluorescence excitation and emission spectra of proliferative cells revealed that fluorescence intensity of malignant cells was significantly less than that of normal cells upon excitation at 290 nm. Fluorescence of both cell lines decreased with decreasing cell concentration, but at each concentration, normal cells had higher fluorescence intensity than malignant cells. Similar differences between the cell lines were observed when brought to quiescence or at stationary phase. Results suggested that the chromophore contributing most significantly to these spectra is tryptophan and its moieties in proteins. This model system demonstrates the specific contribution of H-ras to subcellular chromophores, resulting in a significant difference in their autofluorescence intensity, and implies the potential use of the technique for cancer detection. This model system is potent for analysis of the contribution of other oncogenes and their combinations towards spectral detection of cancer.

Image-resolution enhancement by polyphase FIR Wiener filtering

A method for reconstruction and restoration of super resolution images from sequences of noisy low-resolution images is presented. After estimating the projective transformation parameters between a selected reference image and the observed degraded image sequence frames, the data is rearranged into a sequence with only quantized sub pixel translations. Next, the imaging systems point spread function (PSF) and the auto-correlation function of the image are estimated with a resolution higher than that of the super resolution image. The coefficients of the FIR Wiener filter are computed, low-pass filtered, and decimated so a polyphase filter bank is obtained. Each one of the images in the translated rearranged sequence is filtered by its corresponding polyphase filter. These filtering results are summed and locally normalized according to the apparent data. The super resolution result is refined by estimating the values of pixels that could not be reconstructed by interpolation. The use of the polyphase filters allows exploitation of the input data without any averaging operations needed when implementing conventional FIR Wiener filtering. The presented experimental results show good resolution improvement in presence of noise.

Examining the binding mechanism of 3,4-dihydro-3-(2-oxo-2-phenylethylidene)-quinoxalin-2(1H)-one and its fragments to Cu2+

The structure and stoichiometries of the complexes that could be formed between Cu2+ and 3,4-dihydro-3-(2-oxo-2-phenylethylidene)-quinoxalin-2(1H)-one (1) were investigated by various spectral techniques such as IR, fluorescence, UV–vis and electron paramagnetic resonance. The results suggest that initially 3 : 1 and 2 : 1 (1/Cu2+) complexes are formed at low Cu2+ concentration and upon adding more Cu2+, 1 : 1 (preferred) and 1 : 2 complexes are generated. Since 1 possesses two possible binding sites, further exploration was done by testing the binding ability of Cu2+ to fragments of 1, namely β-enaminoketone derivatives (2–3) and quinoxaline-2-one (4), and by executing calculations of thermodynamic parameters of the reaction between 1 and Cu2+ in ethanol, optimized geometries of the possible complexes, and estimation of stability constants at various stoichiometries. Consequently, a step-by-step binding mechanism is suggested for formation of various complexes between 1 and Cu2+.

FTIR microscopic studies on normal and H-Ras transfected cultured mouse fibroblast cells

Infrared (IR) absorption spectra are well known for their selectivity and minutiae fingerprint of molecular structure. The biochemical changes in the sub-cellular levels developing in abnormal cells, including a majority of cancer forms, manifest themselves in different optical signatures, which can be detected in infrared spectroscopy. The molecular vibrational modes which are responsible for IR absorption spectra, are characteristic of the biochemistry of the cells and their sub-cellular components. We measured the infrared absorption spectra of monolayers of cultured normal and ras gene transformed mouse fibroblasts, using microscopic infrared system (micro-FTIR) technique. The absorption for normal cells was higher than the malignant ones in the spectral range 1000 - 1500 and 2800 - 3000 cm-1. The effect on phospholipid metabolism due to ras gene incorporation is also discussed.

Endogenous fluorescence of normal and malignant fibroblast cultures

Light-induced Fluorescence (LIF) technique is based on fluorescence emitted from intracellular chromophores upon illumination of cells by monochromatic light. We compared LIF emitted from a pair of normal and malignant murine cell lines, differing in H-ras expression. The malignant cells fluoresced significantly less than the normal cells, upon excitation at 290 +/- 10 nm. For both cell types, fluorescence decreased with decreasing cell concentration, but at each concentration, the normal cells fluoresced more than the malignant cells. The effect of viability and metabolic stage of the cells on this pattern was compared. The difference among the cells was not due to a difference in protein or DNA content. Thus, this model system demonstrates the specific contribution of H-ras to sub-cellular chromophores, resulting in a significant difference in their autofluorescence intensity, while measuring both emission and excitation scans. This study suggests a potential use of the LIF technique to distinguish between normal and malignant cells and tissues.

Restoration of an image representing a video sequence recorded under turbulence effects

Turbulence conditions affect video images in tow ways. It causes local blur, and it distorts the geometry of the scene. A video sequence of a still scene recorded under turbulence, appears to contain local random motion of small neighborhoods in the images. The blur is an accumulated results of the imaging point spread function and the local motion. The geometric distortion is due to the fact that small neighborhoods move in different directions. The restoration scheme reported here takes care of the geometric distortion as well as the blur. The geometric distortion is reduced by averaging the gray levels of relatively long video segments. The averaging reduces the geometric distortion, but it increases the blur. The second stage is the estimation of the global point spread function. The blur in the average image is a combination of the effects of the imaging system transfer function,the turbulence, and the averaging of the sequence. The global non-isotropic point spread function is estimated based on edge responses in the average image. A Wiener filter is used for the restoration of the image. The presented experimental result are superior to the results obtained by a previously proposed majority-vote technique.