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Previous works showed that spatially resolved Raman spectra of cytoplasm and nucleus region of single cells exposed to X-rays evidence different features. The present work aims to introduce a new approach to profit from these differences to deeper investigate X-ray irradiation effects on single SH-SY5Y human neuroblastoma cells. For this aim, Raman micro-spectroscopy was performed in vitro on single cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). Spectra from nucleus and cytoplasm regions were selectively acquired. The examination by interval Principal Component Analysis (i-PCA) of the difference spectra obtained by subtracting each cytoplasm-related spectrum from the corresponding one detected at the nucleus enabled us to reveal the subtle modifications of Raman features specific of different spatial cell regions. They were discussed in terms of effects induced by X-ray irradiation on DNA/RNA, lipids, and proteins. The proposed approach enabled us to evidence some features not outlined in previous investigations.

Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined.

Gingival crevicular fluid (GCF) is a site-specific exudate in the gingival sulcus. GCF composition changes in response to diseases or mechanical stimuli, such as those occurring during orthodontic treatments. Raman microspectroscopy (μ-RS) and surface-enhanced Raman spectroscopy (SERS) were adopted for a GCF analysis during different initial phases of orthodontic force application. GCF samples were pooled from informed patients using paper cones. SERS spectra were obtained from GCF extracted from these cones, whereas μ-RS spectra were directly acquired on paper cones without any manipulation. The spectral characteristics of the main functional groups and the changes in cytochrome, amide III, and amide I contributions were highlighted in the different phases of orthodontic treatment with both SERS and μ-RS analysis. μ-RS directly performed on the paper cones together with proper statistical methods can offer an effective approach for the development of a tool for monitoring the processes occurring during orthodontic treatments, which may help the clinician in the choice of type of treatment individually for each patient and accelerate and improve the orthodontic therapy., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

A micro-Raman spectroscopy investigation has been performed in vitro on single human mammary epithelial cells after irradiation by graded x-ray doses. The analysis by principal component analysis (PCA) and interval-PCA (i-PCA) methods has allowed us to point out the small differences in the Raman spectra induced by irradiation. This experimental approach has enabled us to delineate radiation-induced changes in protein, nucleic acid, lipid, and carbohydrate content. In particular, the dose dependence of PCA and i-PCA components has been analyzed. Our results have confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect early molecular changes at the single-cell level following exposure to ionizing radiation. This would help in developing innovative approaches to monitor radiation cancer radiotherapy outcome so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy.

Micro-Raman spectroscopy is a very promising tool for medical applications, thanks to its sensitivity to subtle changes in the chemical and structural characteristics of biological specimens. To fully exploit these promises, building a method of data analysis properly suited for the case under study is crucial. Here, a linear or univariate approach using a R2 determination coefficient is proposed for discriminating Raman spectra even with small differences. The validity of the proposed approach has been tested using Raman spectra of high purity glucose solutions collected in the 600 to 1,600 cm(-1) region and also from solutions with two known solutes at different concentrations. After this validation step, the proposed analysis has been applied to Raman spectra from oral human tissues affected by Pemphigus Vulgaris (PV), a rare life-threatening autoimmune disease, for monitoring disease follow-up. Raman spectra have been obtained in the wavenumber regions from 1,050 to 1,700 cm(-1) and 2,700 to 3,200 cm(-1) from tissues of patients at different stages of pathology (active PV, under therapy and PV in remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage have been detected at 1,150-1,250 cm(-1) (amide III) and 1,420-1,450 cm(-1) (CH3 deformation) regions and around 1,650 cm(-1) (amide I) and 2,930 cm(-1) (CH3 symmetric stretch). The analysis of tissue Raman spectra by the proposed univariate method has allowed us to effectively differentiate tissues at different stages of pathology.

The giant enhancement of Raman signal near silver colloidal nanoparticles is exploited to study the Raman spectrum of Cytochrome c from Saccharomyces cerevisiae (Yeast Cytochrome c--YCc) in the limit of single-molecule. The investigation is performed on proteins both in solution and immobilised onto a glass slide using a quasi resonant laser line as exciting source with low excitation intensity. In both cases, spectra acquired at different times exhibit dramatic temporal fluctuations in both the total spectrum and in the specific line intensity, even though averaging of several individual spectra reproduces the main Raman features of bulk YCc. Analysis of the spectral intensity fluctuations from solutions reveals a multimodal distribution of some specific Raman lines, consistent with the approaching of single molecule regime. Among other results, the statistical analysis of the spectra from immobilised samples seems to indicate dynamical processes involving the reorientational of the heme with respect to the metal surface.