Biomedical Spectroscopy and Imaging

Experimental and theoretical investigations have been carried out on the interactions of urea and, as a comparison, water with phospholipids. Firstly, Langmuir monolayer technique demonstrated that the presence of urea in the subphase caused a lateral expansion of the molecular monolayer of dipalmitoylphosphatidylcholine (DPPC) and a number of other phospholipids. This is attributed to possible direct interaction between urea and the lipids and thus insertion of urea into the head group region. Secondly, Raman spectra showed that the N–H stretching bands of urea shift to lower wavenumber while the symmetric stretching mode of PO₂ of lipids shift to higher wavenumber, when urea coexists with DPPC in aqueous codispersions. This is consistent with the formation of hydrogen bonds between urea and DPPC molecule. Thirdly, quantum chemical calculations of the first principle demonstrated that the interaction of urea with lipid's amino group, phosphate group, and glycerol backbone are significantly stronger than that with water. Hence it is concluded that urea can interact specifically with the headgroup of phospholipids in an aqueous solution. The results would be helpful to understand the effect of urea on biomembranes and other biomolecular structures.

G protein-coupled receptors (GPCR) are integral membrane proteins that transmit signals from external stimuli to the cell interior via activation of GTP-binding proteins (G proteins) thereby mediating key sensorial, hormonal, metabolic, immunological and neurotransmission processes. Elucidating their structure and mechanism of interaction with extracellular and intracellular binding partners is of fundamental importance and highly relevant to rational design of new effective drugs. Surface plasmon resonance (SPR) has become a method of choice for studying biomolecular interactions at interfaces because measurements take place in real-time and do not require labeling of any of the interactants. However, due to the particular challenges imposed by the high hydrophobicity of membrane proteins and the great diversity of receptor-stimulating ligands, the application of this technique to characterize interactions of GPCR is still in the developmental phase. Here we give an overview of the principle of SPR and analyze current approaches for the preparation of the sensor chip surface, capture and stabilization of GPCR, and experimental design to characterize their interaction with ligands, G proteins and specific antibodies.

BACKGROUND: The SERS imaging of the intracellular microenvironment may provide valuable insight into the chemical composition and distribution of a cellular biological system.

OBJECTIVE: In order to develop the SERS-based methodology for the cellular uptake of gold nanoparticles conjugated with the fluorescent dye (rhodamine 6G) was investigated in endothelial EA. hy. 926 cell line. A few timeframes were chosen to assess an effect of particles accessibility on their distribution in the cells.

METHODS: Confocal Raman imaging was employed to evaluate the distribution of SERS response within the cells incubated with labelled Au nanoparticles. A cluster K-means analysis was used in the construction of SERS maps.

RESULTS: The cellular uptake of the gold nanoparticles labelled with rhodamine 6G occured up to 12 h of the incubation. Along with the SERS signal of the dye, the bands originating mainly from aromatic amino acid residues and nucleobases were found in the spectra.

CONCLUSIONS: The optimal accumulation of label-nanoparticles is found for up to 6 h incubation of the particles, confocal SERS imaging of labelled endothelial cells can serve as a powerful tool for further examination of the physiological and pathological changes in endothelial intracellular microenvironment.

The vascular wall cross-section was submitted to multiparametric analysis with the application of several micro-scale methods: Raman and atomic force microscopy (AFM) imaging as well as immunohistochemical (IHC) staining. Both IHC staining as a standard reference method and Raman imaging allowed for visualization of tissue components such as elastin fibers or cell nuclei, moreover, Raman spectroscopy provided specific biochemical information about sample composition. Application of AFM complemented the chemical information with the data about the topography of the studied sample. Developing such a methodology allows for the multiparameter description of the vessel wall status that could be useful to monitor ex vivo the disease progression and effects of treatment in experimental diabetes, atherosclerosis or hypertension.

We report the results of a clinical study to characterize the inter-anatomical variability of in vivo Raman spectra of normal oral cavity of healthy volunteers, and investigate its effect on the outcome of statistical discrimination of malignant and potentially malignant oral lesions from the healthy oral mucosa. An unsupervised cluster analysis using Fuzzy c-means clustering algorithm was conducted for quantifying the underlying structure of the normal oral tissue spectra. The algorithm was found to segment the normal oral tissue sites, based on similarity of spectral patterns, into four major anatomical clusters (AC): (1) outer lip, and lip vermillion border into AC-I with an accuracy of 80%; (2) buccal mucosa into AC-II with an accuracy of 72%; (3) hard palate into AC-III with an accuracy of 92%; (4) dorsal, lateral and ventral tongue and soft palate into AC-IV with an accuracy of 76%. A probabilistic multi-class diagnostic algorithm, developed for supervised classification, was used to classify the whole set of measured tissue Raman spectra into three categories: normal, potentially malignant and malignant. The results showed that the diagnostic algorithm, when applied on the pooled set of spectra from all the clusters, correctly discriminated normal, malignant and potentially malignant tissue sites with 86%, 88% and 86% accuracy respectively, which amounts to an overall accuracy of 87%. However, when the anatomy-matched data sets were considered, the overall classification accuracy was found to improve to 95% with the algorithm correctly discriminating the corresponding tissue sites with 94%, 99% and 91% accuracy respectively.

BACKGROUND: The amount of UV rays that reach the ground as well as the UVA/UVB ratio depend on several factors such as latitude, season, cloud coverage and time of day, both UVA and UVB radiation are responsible of a great variety of skin disorders such as actinic keratoses, non-melanoma skin cancer and melanoma.

METHODS: In this work the solar radiation of face and chest is calculated for 80 Mexican-mestizo volunteers using their melanin concentration relative to a non-photoexposed area which were obtained using diffuse reflectance spectrophotometry.

RESULTS: The results show that the points of highest solar exposure are cheeks and cleavage, the points with lowest solar exposure are the chin and lower chest. It was also observed that the amount of solar exposure influenced the clinical assessment of skin phototype.

CONCLUSION: The results support the concept that proper education on solar preventive measures mainly on the head and cleavage is fundamental to reduce the incidence of photaging and skin cancer, and that diffuse reflectance spectrophotometry can be used to reduce the effect of chronic sun exposure in the determination of the skin phototype.

The relationship between arsenic (As) and selenium (Se) in the human body is poorly understood. We have investigated the concentrations of urinary As and Se in three ethnic groups (n=63) in the United Kingdom and show that there is a positive correlation (r=0.62, p<0.001) between total concentrations of As and Se and that the ratio of these two elements is stable, with a mean value (±SD) of 0.7±0.4. Furthermore, concentrations of individual arsenic species methylarsonate (MA), dimethylarsinate (DMA) and arsenobetaine (AB) in the urine samples show a positive correlation with total Se (As(III) and As(V) were not detected). The intra-individual variation of the As:Se ratio also remains stable over time, as determined by monitoring a volunteer over a period of one year, and deviates only after recent seafood consumption. It appears that the ratio is also stable across diverse populations across different cultures and continents, evident from our calculation of As:Se ratio from concentrations of these elements found in urine samples from different populations published in the literature. Our study involved analysis of 63 urine samples from three ethnic groups (White Caucasian n=20, Asian n=21 and Somali n=22), 58 urine samples from 29 Ramadan fasting volunteers and 12 from one volunteer whose urine samples were collected over a period of one year. All the participants completed a lifestyle questionnaire and were asked to refrain from eating seafood or fish for three days prior to collection of the sample. Total As and Se in urine were determined by inductively coupled plasma mass spectrometry (ICP-MS). As species (AB, DMA, MA, As(III), As(V)) were determined by using high performance liquid chromatography (HPLC) combined with ICP-MS. Mean ± SD As:Se ratios of 0.8±0.4, 0.7±0.4, 0.4±0.2, 0.7±0.3 and 1.2±0.3 were obtained for the Asian, White Caucasian, Somali, fasting, and one volunteer respectively, giving an overall mean of 0.7±0.4 (SD). It is noteworthy, that when comparing ethnic differences, the Somali group shows a statistically significant lower As:Se ratio (0.4±0.2, p<0.05) compared to Asian and White Caucasian groups; this is ascribed to lower urinary As concentrations in this group. The study over one year with a single volunteer revealed that recent (within 3 days) seafood consumption results in a significantly different (p<0.05) As:Se ratio (4.0). We have calculated from the literature the value of As:Se for populations, exposed to As through drinking water, can range from 2.0–9.6. Based on our own work and the values we calculated from other studies we suggest that the baseline range for mean As:Se ratio is 0.4–1.2, provided that the urine samples are collected in the absence of recent consumption of seafood. The relatively stable As:Se ratio observed in this study suggests a link between these two elements in humans adding support to earlier studies with animals and humans exposed to inorganic arsenic in drinking water which reported interaction between these elements and that Se may play a role in counteracting As toxicity.