Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint sig-nals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantita-tive applications of SERS in various fields and several SERS methods have been implemented for each specific applica-tion, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in high-ly-regulated environments is still considered risky and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlabora-tory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quan-tification with SERS arose within the framework of Working Group 1 (WG1) of the COST Action BM1401 Ra-man4Clinics32 in an effort to overcome the problematic perception of quantitative SERS methods. Here we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 41 researchers. In this study we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method, and to compare differ-ent methods. In our opinion, this is a first important step toward a “standardization” process of SERS protocols, not proposed by a single laboratory but by a larger community.

The intracellular pH plays an important role in various cellular processes.

Spectroscopy-based approaches can provide an insight into the biochemical composition of a tissue sample. In the present work Fourier transform infrared (FT-IR) spectroscopy was used to develop a reliable methodology to study the content of free fatty acids, triglycerides, cholesteryl esters as well as cholesterol in aorta from mice with atherosclerosis (ApoE/LDLR^−/− mice). In particular, distribution and concentration of palmitic, oleic and linoleic acid derivatives were analyzed. Spectral analysis of pure compounds allowed for clear discrimination between free fatty acids and other similar moieties based on the carbonyl band position (1699–1710 cm⁻¹ range). In order to distinguish cholesteryl esters from triglycerides a ratio of carbonyl band to signal at 1010 cm⁻¹ was used. Imaging of lipids in atherosclerotic aortic lesions in ApoE/LDLR^−/− mice was followed by Hierarchical Cluster Analysis (HCA). The aorta from C57Bl/6J control mice (fed with chow diet) was used for comparison. The measurements were completed with an FT-IR spectrometer equipped with a 128 × 128 FPA detector. In cross-section of aorta from ApoE/LDLR^−/− mice a region of atherosclerotic plaque was clearly identified by HCA, which was later divided into 2 sub-regions, one characterized by the higher content of cholesterol, while the other by higher contents of cholesteryl esters. HCA of tissues deposited on normal microscopic glass, hence limited to the 2200–3800 cm⁻¹ spectral range, also identified a region of atherosclerotic plaque. Importantly, this region correlates with the area stained by standard histological staining for atherosclerotic plaque (Oil Red O). In conclusion, the use of FT-IR and HCA may provide a novel tool for qualitative and quantitative analysis of contents and distribution of lipids in atherosclerotic plaque.