Biomedical Spectroscopy and Imaging - Volume 3, issue 3
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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: Amylin is a small polypeptide that is implicated in type 2 diabetes. This article reviews the data and conclusions presented at the 2013 ECSBM meeting in Oxford about the aggregation mechanism of amylin into toxic oligomers and fibers. The process was studied using a pulse shaping method for collecting two-dimensional infrared (2D IR) spectra in order to monitor aggregation in real time. Isotope labeling was used to measure the secondary structure content of individual residues. It was found that prior to fiber formation, peptides aggregate into an intermediate that has parallel β-structure from residues 23–27, called the “FGAIL” region due…to its sequence. This region has long been implicated in the aggregation process, but with confusing and often contradictory experimental results. Our work using 2D IR spectroscopy has helped reconcile these perceived contradictions and provides a mechanism that helps explain sequence homology between species, drug inhibition, and aggregation kinetics.
Keywords: Amylin, infrared spectroscopy, 2D IR, aggregation pathway, intermediate
Abstract: Raman spectroscopy has shown considerable promise as a medical diagnostic tool. The technique is safe for use in vivo and is capable of rapid, objective assessment of neoplastic and inflammatory tissue enabling early diagnosis and targeted treatment. We review potential applications of Raman spectroscopy in the oesophagus, colon and breast where this new technology has the potential to revolutionise patient care.
Abstract: Cryopreservation of bacteria requires specific cells pre-adaptation as well as controlled freezing protocol to minimize membrane damage resulting from osmotic cell dehydration. Membrane fluidity has an important role in biophysical events taking place at subzero temperatures, by facilitating or not the exchanges between intracellular and extracellular media. An original approach using Synchrotron fluorescence polarization microscopy was developed to measure membrane fluidity of single bacteria. Cells were labelled with the cytoplasmic membrane probe trimethylammoniumdiphenylhexatriene (TMA-DPH). The degrees of liberty of TMA-DPH inside the bilayer provide a direct marker of membrane fluidity under deep UV excitation. Couples of fluorescence polarization images from…zero to forty degrees were recorded by inserting polarizers into the excitation and emission paths. The fluorescence anisotropy of the images was calculated thus allowing the assessment of the cell membrane fluidity. Results indicate intercellular and intracellular heterogeneities with subdomains appearing at low temperature for freeze-sensitive cells.
Keywords: Anisotropy, lactic acid bacteria, membrane, fluidity, temperature effect, DUV
Abstract: Fluorescence lifetime imaging has been used to observe the real-time uptake in live mammalian cells of a combretastatin-type drug analogue that is a substituted stilbene with a cyano group at the bridging olefinic bond. Fluorescence spectra in a range of solvents show that this molecule has a substantial increase in dipole moment on promotion to the first excited singlet state that is indicative of intramolecular charge transfer (CT) from the methoxy substituents on the aromatic rings to the electron-withdrawing cyano group. This CT process is also demonstrated in picosecond time-resolved infrared measurements. Consistent with these observations, the molecule has been…found to have a large two-photon absorption cross-section, and suggests potential routes to the design of a molecule that could be effectively activated by photoisomerisation in a two-photon process.
Abstract: Terahertz time-domain, FTIR and Raman spectroscopic techniques are used to study the interaction of chymotrypsin (CT) with 18-crown-6 (CE). The THz and FTIR spectra of CT demonstrate only three weak spectral lines whereas the spectra of the CT–CE mixtures exhibit several developed spectral features. Raman spectroscopy proves the presence of the bands in the low-frequency range. The results indicate that an increase in the functional activity of CT in nonaqueous solvents can be related to the interaction of the protonated amino groups of the protein with the CE molecules. FTIR and Raman spectra of lyophilized native and denatured protein are…compared in the fingerprint and low-frequency ranges. Possible structural differences related to the spectral changes are discussed.
Abstract: Raman spectroscopy is a powerful analytical tool however it suffers from poor sensitivity. Surface Enhanced Raman Spectroscopy (SERS) takes advantage of localised surface plasmons (LSPs) generated by metal nanostructures to significantly improve the sensitivity of Raman spectroscopy [J. Raman Spec. 43 (2012), 380–388], though this often results in poor reproducibility. Using silver colloids synthesised by the method outlined by Leopold and Lendl [Journal of Physical Chemistry B 107 (2003), 5723–5727], we have sought to improve both the sensitivity and reproducibility of the SERS spectra produced by this popular nanoparticle synthesis method.
Abstract: The enhanced green fluorescent protein (EGFP) is broadly used as an intracellular fluorescent marker. Its numerous applications require a comprehensive understanding of EGFP folding properties, as well as the properties of EGFP-derived mutants. Herein, we report on the spectroscopic characterization of two single-cysteine mutants, C48S-EGFP and C70S-EGFP, which were prepared to monitor the protein folding dynamics by EPR, specifically in the close vicinity of both cysteine residues. Chromophore absorption, excitation and emission spectra for these mutants were similar to those for EGFP. Both single-cysteine mutants were more susceptible to aggregation than EGFP in E. coli. C48S-EGFP exhibited similar…resistance to chemical denaturation as EGFP. In contrast, C70S-EGFP was less resistant to denaturation in guanidine hydrochloride and its folding was less efficient. Hence, C48S-EGFP seems to be a more suitable probe for EPR measurements than C70S-EGFP.
Keywords: Enhanced green fluorescent protein (EGFP), C48S-EGFP, C70S-EGFP, spectroscopic properties, fluorescence, absorption
Abstract: BACKGROUND: Biomechanical and biochemical properties are altered between healthy, cancerous, and metastatic cancer cells. These changes include altered lipid metabolism, resulting in varied lipid contents; varied nuclear organisation and size, which affect proliferation and transcription; and lower adhesion and rigidity, which enhance their capacities to escape primary tumour site, invade local stroma and metastasise. OBJECTIVE: In the literature, the mechanical and the chemical properties have been obtained from cells separately. We wish to demonstrate the techniques can be combined and applied to the same cells. This will allow for the correlation between chemical and mechanical properties to be studied,…and therefore the gaining of knowledge on how the underlying chemistry affects the cell's mechanical properties. Such information has implications on a better understanding of tumour progression. METHODS: 3D confocal Raman and atomic force microscopy (AFM) imaging – which are correlative and complementary – were applied to the same U-87MG glioma cells to describe their organelles' chemistry and volumes and the cells' rigidity via Young's modulus (YM) calculation. RESULTS: Chemical information and the YM were successfully obtained from the same cell using the described combined techniques. Using principal component analysis (PCA), the cells were found to differ in their lipid composition (in particular, cholesterol ester) even though they were from the same culture. This strongly implies the need of single cell analysis. CONCLUSION: The combined techniques allowed for the extraction of biomechanical and biochemical information from the same cancer cells. This will enable the investigation of their correlation on the single cell level. The combined techniques can provide powerful insights that will lead to improvements in brain cancer treatments, preventative measures and provide targets for the effectiveness of treatment.
Keywords: Raman, AFM, metastases, 3D imaging, cancer, brain
Abstract: Elastic fibers are responsible for elasticity of organs and tissues in vertebrates. Elastin is the amorphous component of these complex biopolymers and is an insoluble, highly cross-linked, hydrophobic protein assembled from a soluble precursor called tropoelastin. In order to elucidate the detailed structure of human tropoelastin, the exon-by-exon chemical synthesis of all the polypeptide sequences of the protein was accomplished. Herein, we review some of the most relevant results obtained at molecular, by using CD and NMR spectroscopies, and supramolecular level mainly by TEM. The overall results obtained for each polypeptide let us to assembly the elastin puzzle and envisaging…the whole structure of human tropoelastin. Analogous structural studies were carried out on some polypeptide sequences inspired to elastomeric proteins. These findings give useful insights into the elasticity mechanism of all elastomeric proteins. The knowledge can be exploited in the near future to design novel bioelastomers with tailored material properties for use in medicine and industry.
Keywords: Elastin, amyloid-like, polyproline II, transmission electron microscopy, circular dichroism, nuclear magnetic resonance
Abstract: Infrared absorption spectra of carbon monoxide molecule coordinated by the heme iron of carbonmonoxy heme proteins are widely used to study their structure and dynamics. In this paper we use results of X-ray study of carbonmonoxy myoglobin to elucidate the structures of spectroscopically observed conformational substates of this protein. It is shown that A3 substate corresponds to the structure with water molecule hydrogen bonded to the distal histidine, whereas in the A1 conformation this molecule is absent. We also show that redistribution of electronic density of the distal histidine and the water molecule as a result of their…interaction must be taken into account when predicting their positions in the heme pocket.
Keywords: Myoglobin, infrared, protein dynamics, conformational substates
Abstract: BACKGROUND: The rabbit Vx2 liver tumor is a fast-growing carcinoma model commonly used to study tumor behaviours under cancer treatments. The reduction of tumor viability and the degree of induced necrosis are the common criteria to evaluate the efficacy of cancer treatments. Currently, it is not easy to perform a rapid and reproducible tissue quantification by histopathological analysis. OBJECTIVE: Our objective was to use infrared-imaging combined with linear-discriminant-analysis model (LDA) to automatically quantify the necrotic effect induced by doxorubicin-eluting-implants (DEI) on a Vx2 liver tumors model. METHODS: Three rabbits were subjected to DEI treatment and compared to a…control group (CTRL) of nine rabbits. Tumor bearing livers were resected, fixed in formalin and embedded in paraffin. On two consecutive sections, we performed hematoxylin–eosin–saffron (HES) staining and infrared-imaging. Infrared-images were then subjected to the LDA-model analysis. RESULTS: The LDA and HES images strongly correlated for viable and necrotic tumor tissues. For the DEI group, the model determined that the surface of viable tumor represented 0.24±5% of the tumor (CTRL: 55.71±17%, P<0.0001) and the necrotic tissue 66.46±20% of the tumor (CTRL: 18.45±12%, P<0.0001). CONCLUSIONS: Our results showed that infrared-imaging coupled with LDA-model analysis could be a helpful to easily and objectively assess the treatment efficacy.
Abstract: This work brings together previous work on catechol (1,2-dihydroxybenzene) and resorcinol (1,3-dihydroxybenzene) with recent results from experiments on hydroquinone (1,4-dihydroxybenzene). The experiments utilise time-resolved ion yield and velocity map imaging techniques to demonstrate how the position of the OH functional groups influences the excited state dynamics of these molecules. The mechanisms responsible for these dynamics are explored, with the focus on H-atom tunnelling mediated photodissociation. In all cases, tunnelling occurs beneath a conical intersection formed between the excited 11 ππ* state and the 11 πσ* state, which is dissociative along the O–H stretch coordinate. Tunnelling beneath this barrier yields high…kinetic energy (~5000–7000 cm−1 ) H-atoms. The excited state lifetime of these molecules ranges from hundreds of femtoseconds to several nanoseconds and is found to be dependent on excitation energy as well as the relative positions of the OH functional groups.
Abstract: BACKGROUND: During pathological and/or physiological processes, such as tumoral progression or aging, human skin undergoes molecular and structural changes mainly due to alterations of dermal structural proteins such as type I collagen. These modifications in dermis are probably due to collagen fibers rearrangement and reorientation which have not been thoroughly studied up to now. FT-IR (Fourier transform infrared) microspectroscopy associated to polarization measurement appears as an interesting method to determine in situ the alignment of type I collagen fibers. OBJECTIVE: In this paper, two different clustering algorithms were applied to determine different layers in human skin. Besides, we used…polarized FT-IR imaging to evaluate the molecular organization of dermal collagen. METHODS: To do this, a cryosection of skin from 48-year-old woman was analyzed. Acquired FT-IR images were firstly processed using K-means and fuzzy C-means (FCM). FCM was chosen on polarized FT-IR images in order to highlight the molecular orientation of dermal collagen fibrils. The ratio of Amide I/Amide II bands integrated intensities was computed in order to assess the orientation of collagen fibrils. RESULTS AND CONCLUSION: This methodology permitted to reveal the potential of polarized FT-IR microspectroscopy for the characterization of type I collagen network in human skin. Our approach could help to find innovative applications in dermatology as well as in cosmetics.
Keywords: Polarized FT-IR imaging, type I collagen, collagen orientation, clustering methods
Abstract: Disulfide bridges of bovine α-thrombin are studied using the analysis of the Raman spectral features in the spectral interval 500–550 cm−1 . The changes under study are caused by the transitions from lyophilized state to the native solution in PBS with a protein concentration of 2.8 mM (100 mg/ml) and from native solution to solution with partially reduced disulfide bridges. The reduction takes place when dithiothreitol (DTT) is added to the sample.
Abstract: BACKGROUND: The techniques of second harmonic generation (SHG) and two-photon excitation of fluorescence microscopy (TPEFM) have shown as powerful tools to investigate collagen and extracellular matrix components. OBJECTIVE: The aim of this study is evaluate the feasibility of using femtosecond lasers of high intensity as an auxiliary treatment of burn patients using an in vivo model, monitoring and characterizing the healing process by histology and SHG+TPEFM. METHODS: Samples from three dorsum areas of Wistar rats anaesthetized were burned by water vapour exposure and treated by debridement and laser ablation at the third day post burned. To differentiating the…healing process, skin fresh biopsies at four different days of study were evaluated by histology, TPEFM and SHG. RESULTS: Changes in the skin caused by vapour exposure were observed by histological images and characterized by TPEFM+SHG images. The healing process of burned skin was observed by regeneration of its morphology comparing histological images. The integrity of collagen and components of extracellular matrix are evidence of a normal skin detected by TPEFM+SHG. CONCLUSIONS: The methods used to characterize the tissue are useful to validate the femtosecond lasers ablation treatment of burned skin.
Abstract: The use of lasers on bone cut can provide a series of advantages for both the surgeon and the patient. However, for a safe and efficient application it is necessary to know the exact effects that the laser causes in the bone tissue. The aim of this work was to study the effects of Er,Cr:YSGG irradiation in bone tissue using the ATR-FTIR technique. Pieces of tibia rabbit bone were divided in six groups with three samples per group. In one of the groups the samples did not undergo any treatment; in the others the samples were laser irradiated with different…energy densities. The infrared spectra acquisition was made using an ATR accessory. For a semi-quantitative analysis, the area under each band was calculated and normalized by the phosphate band area of the same spectrum. The results showed a gradual material loss as the energy density increased in the bands of water, amide I, and carbonate, amide II, amide II and collagen. This is probably caused by the temperature rise due to laser irradiation. These results are the first steps in testing the Er,Cr:YSGG laser efficacy as a cutting tool, a pivotal aspect of its consolidation in clinical procedures.