Biomedical Spectroscopy and Imaging - Volume 5, issue 2
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This journal has been discontinued. Volume 10 was the last complete volume ofBiomedical Spectroscopy and Imaging.
Biomedical Spectroscopy and Imaging (BSI) is a multidisciplinary journal devoted to the timely publication of basic and applied research that uses spectroscopic and imaging techniques in different areas of life science including biology, biochemistry, biotechnology, bionanotechnology, environmental science, food science, pharmaceutical science, physiology and medicine. Scientists are encouraged to submit their work for publication in the form of original articles, brief communications, rapid communications, reviews and mini-reviews.
The journal is dedicated to providing a single forum for experts in spectroscopy and imaging as applied to biomedical problems, and also for life scientists who use these powerful methods for advancing their research work. BSI aims to promote communication, understanding and synergy across the diverse disciplines that rely on spectroscopy and imaging. It also encourages the submission of articles describing development of new devices and technologies, based on spectroscopy and imaging methods, for application in diverse areas including medicine, biomedical science, biomaterials science, environmental science, pharmaceutical science, proteomics, genomics, metabolomics, microbiology, biotechnology, genetic engineering, nanotechnology, etc.
Abstract: This review summarises our infrared spectroscopy and density functional theory studies on the mutual interactions between enzymes and their substrates. We investigated phosphoenolpyruvate bound to pyruvate kinase (EC 2.7.1.40, M1 isozyme), ATP bound to the Ca2+ -ATPase (SERCA1a), and the aspartylphosphate moiety of the Ca2+ -ATPase phosphoenzyme E2P. Conformational changes of the enzymes and distortions of substrate structure are discussed. In all cases, the infrared absorption of the substrate in the enzyme environment could be identified by a combination of reaction-induced difference spectroscopy and isotopic labelling. The experimentally-determined vibrational frequencies were interpreted in structural terms using experimental correlations or modelling…of the active site in density functional theory calculations. For none of the three systems, a weakening of the bond that is cleaved in the following enzymatic reaction could be detected in the ground state of the enzyme-substrate complex. However, for the dephosphorylation reaction of the Ca2+ -ATPase phosphoenzyme E2P, a high energy intermediate, not detected in experiments, is the reactant state according to density functional theory calculations.
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Abstract: Typical commercial Raman micro-spectroscopic systems do not offer much flexibility to the end user, thus limiting potential research applications. We present a design of a simple, highly flexible and portable confocal Raman microscope with a detailed list of parts. The system can perform spectral acquisition in different modes: single-point spectroscopy, hyperspectral point mapping or hyperspectral line mapping. Moreover, the microscope can be easily converted between inverted and upright configurations, which can be beneficial for specific situations. Fiber coupling enables to connect various lasers for excitation and spectrometer/CCD combinations for signal detection. The performance of the instrument is demonstrated via Raman…spectroscopy at 785 nm excitation wavelength, single point mapping of pancreatic cancer cells placed onto a quartz substrate and line mapping of polystyrene beads.
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Abstract: Background: Lung cancer is the leading cause of death for male and female cancer patients alike. Early diagnosis improves prognosis. A blood test would be a valuable support. Objective: Infrared spectroscopy provides a label-free biochemical fingerprint of a sample. A study was conducted on 161 patients with initial cancer suspicion to identify and verify spectral biomarker candidate patterns to detect non-small cell lung carcinoma (NSCLC). Methods: Blood serum and plasma samples were analysed with an automated FTIR spectroscopic system. Two pattern recognition algorithms and two classifiers were applied. Monte Carlo cross validation was performed with linear…discriminant analysis and random forest classification. Results: Marker patterns for the discrimination of cancer from clinically relevant disease control patients were identified in FTIR spectra of blood samples. An accuracy of up to 79% was achieved. Squamous cell and adenocarcinoma patients were separable with an accuracy of 80%. Conclusions: The study demonstrates the applicability of FTIR spectroscopic blood testing for lung cancer detection. Evidence for cancer subtype discrimination is given. With an improved performance, the method could be developed as a routine diagnostic tool for blood testing detecting NSCLC.
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Abstract: BACKGROUND: Proteins are sensitive to environmental conditions. Whether they are produced for therapeutic purposes or for fundamental research, the integrity of their structure and post-traductional modifications are key issues. Measuring glycosylation or phosphorylation level as well as their secondary structure most often rely on complex and indirect experiments. Infrared spectroscopy presents a series of advantages related to its multivariate character. There is a lack of high-throughput methods able to analyse these parameters. OBJECTIVE: In this paper we attempted to combine protein microarrays and infrared imaging for high throughput analysis of proteins. METHODS: A protein microarrayer was…used to produce protein microarrays on BaF2 slides transparent in the mid-infrared. Spot density was about 25 spots/mm2 . A 128 × 128 focal plane array infrared detector was used to record images of the protein microarrays. RESULTS: We show that 100 µm diameter spot are easily analyzed. Spots obtained with low protein concentrations, resulting in an average of a single protein monolayer (ca 3 fg/µm2 for a 66 kDa protein) provided good quality spectra. CONCLUSIONS: Infrared imaging is a label free, high throughput method, able to analyse protein microarrays and to take advantage from the wide information available in the infrared spectra.
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Keywords: Protein microarray, infrared imaging, FTIR, protein structure, high throughput
Abstract: BACKGROUND: It is now recognized that tumor cells can actively alter their microenvironment and that this remodelled microenvironment can subsequently play a critical role in cancer progression and influence therapeutic responses. To date, the molecular heterogeneity within a 3D cancer cell colony and its influence on the extracellular matrix have not been studied by infrared imaging. OBJECTIVE: The objective of this study is to investigate by mid-infrared imaging 3D Matrigel-embedded colonies of pure MCF-7 human mammary adenocarcinoma cell line and the surrounding microenvironment after undergoing formalin fixation and paraffin embedding (FFPE). METHODS: In order to better…reproduce the procedure used for preservation and storage of clinical tissue specimens for pathologic analysis, 7- and 10-day MCF-7 colonies embedded and grown in Matrigel were FFPE-treated; 4-µm-thick sections were cut, mounted on barium fluoride window and deparaffinized. The Fourier transform infrared (FTIR) images of 4096 spectra were collected in transmission mode using a FPA-based FTIR imaging system. They were pre-processed and analysed by principal component and K-mean cluster analyses. RESULTS: At 1654 cm−1 and 1234 cm−1 , the intensity of absorption in the colonies is significantly higher than in Matrigel. It can be also noted, on the one hand, that there is a spectral heterogeneity in the intracolonial distribution of the absorbances at 1654, 1644, 1640 and 1634 cm−1 (Amide I range) possibly due to changes in protein secondary structures. On the other hand, we observe that Matrigel close to MCF-7 colonies appears altered with respect to more distant Matrigel matrix. CONCLUSIONS: FTIR imaging allowed us to highlight changes in the chemical content in MCF-7 colonies and their direct vicinity in Matrigel-embedded 3D cultures.
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Keywords: FTIR imaging, 3D cell culture, extracellular matrix
Abstract: The membrane plays an important role in the structure and function of membrane proteins. We studied the influence of the lipid environment on the photocycle of the membrane protein bacteriorhodopsin (BR) with time-resolved step-scan FTIR spectroscopy. Proton transfer dynamics was monitored with microsecond time resolution for BR embedded in the native purple membrane as well as reconstituted into DOPC liposomes. We observed altered protonation dynamics of the Schiff base and the primary proton acceptor Asp85, revealing a faster rise as well as decay of the M state for BR surrounded by DOPC lipids. The purple membrane consists of a lipid…composition that adapts better to the protein shape resulting in a stronger protein-membrane interaction as compared to the uniform DOPC lipid environment. Conformational dynamics and the correlated protonation dynamics are affected by the altered protein-membrane interaction explaining the faster photoreaction of BR in DOPC liposomes. Here we demonstrate the high sensitivity of the proton transfer dynamics to the lipid environment of BR.
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Abstract: Background: Red blood cells (RBC) are obtained by non-invasive methods and widely used for diagnostic tests of health status. Hyperspectral Dark Field Microscopy (HDFM) is a promising technique for nanoscale bio imaging and spectral analysis without additional sample preparation. Objective: Develop a protocol for human RBC characterization by HDFM, checking the feasibility of a reference spectral library that can image and afford a new comprehensive descriptor of RBC status. Method: A step-by-step protocol for HDFM measurement of human RBC was for the first time established using 5 µl of EDTA-treated whole blood from healthy adults (n =…30 ). Hyperspectral characteristics of solutions/suspensions at biological concentrations of phospholipids, hemoglobin, spectrin, cholesterol and protoporphyrin IX, as the most relevant RBC components, were also determined. Results: A library made of 8 end-member spectra and classification of their spectral distribution carried out by Single Angle Mapper (SAM) were determined, furnishing a comprehensive mapping and descriptor of healthy human RBC. The spectra of single components allowed some of the RBC spectral bands to be attributed. Conclusions: This work reports for the first time the hyperspectral optical imaging of the human RBC by a library made of 8 scattering spectra, whose spectral signatures are compared with those of the main RBC molecular components. The percent distribution of the spectral end-members was also achieved, thus giving for the first time the HDFM mapping of human healthy RBCs. The protocol developed herein allows the clinical potential of hyperspectral imaging to be developed for the use of RBC mapping in health and disease.
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Abstract: BACKGROUND: In IR difference spectroscopy of proteins and biological systems, as in many other biophysical techniques, experiments at different hydration levels, and after H/D or H2 16 O/H2 18 O exchange are particularly important, for band assignment and for mechanistic studies. OBJECTIVE: Evaluate the kinetics, the effectiveness and the feasibility of an isopiestic approach for controlled hydration and isotopic exchange on bacterial photosynthetic reaction centers as a model for membrane proteins. METHODS: Mid-Infrared and Near Infrared spectroscopy experiments at different times and kinetic data treatment. RESULTS: Equilibria for hydration/dehydration, and for isotopic exchange are…rapid (hour time scale) and can easily be applied in normal conditions. The exact hydration state and the effectiveness of isotopic exchange can be precisely assessed through FTIR or NIR spectroscopies. Furthermore, effectiveness of isotopic exchange (especially for internal exchangeable residues or molecules) appears to be higher compared to traditional methods. CONCLUSIONS: The isopiestic method can be easily applied to obtain controlled hydration conditions and high rate of isotopic exchange. This approach can be in principle applied to any biophysical technique.
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Abstract: BACKGROUND: Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies, where the protein deposition occurs, and in the biotechnology field like the food technology where many processes in food manufacturing are based on thermal treatments. OBJECTIVE: The influence of Cu2+ or Zn2+ ions on the thermal aggregation process of Bovine β -lactoglobulin (BLG) and Bovine Serum Albumin (BSA), two protein models, was studied with the aim of delineating the role of these ions in the protein aggregation kinetics and to clarify the related molecular mechanisms. METHODS: The protein structure changes were…monitored by Raman spectroscopy, whereas the aggregate growth was followed by Dynamic Light Scattering measurements. RESULTS: Both metal ions are able to favour the BLG aggregation, whereas only Zn2+ ions have a promoter effect on the thermal aggregation of BSA. The reason of this different behaviour is that the BLG aggregation evolution is manly affected by the redistribution of charges, whereas that of BSA by the metal coordination binding which depends on metal. CONCLUSIONS: Raman spectroscopy, combined with dynamic light scattering experiments, was very useful in identifying the role played by Cu2+ and Zn2+ on the aggregation pathways of BLG and BSA. The results provide evidence for the role of histidine residues both in the redistribution of charges and in the two modes of metal binding that take place in BLG- and BSA-containing systems, respectively.
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Keywords: Beta-lactoglobulin, Bovine Serum Albumin (BSA), copper and zinc ions, Raman spectroscopy, dynamic light scattering