Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Biomolecular composition of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination.

The study aimed to analyze changes in biomolecular composition of granulosa and theca interna cells of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination. Medium antral follicles (n = 4/each group) were cultured alone (C; control) or in the presence of 1α,25(OH)2D3 (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination, VD and I (VD+I). Then paraplast-embedded ovarian follicles were used for Fourier Transform Infrared (FTIR) spectroscopy and respective histological stainings. FTIR analysis revealed changes in the content of fibrous proteins (mainly collagens) within theca interna following vitamin D3 and insulin co-administration that was verified by Masson's trichrome staining. Treatment-dependent differences were also observed in the secondary structure of proteins, indicating enhanced conversion to α-helices in response to vitamin D3 and insulin action/interaction in both follicular compartments. In the granulosa and theca interna layers, tendency to lower DNA content in the VD+I group was noted and confirmed by Fulgen's staining. Finally, altered monosaccharides production in both follicular layers was found. Based on FTIR results, it is possible to attribute the observed alterations to biological processes that could be regulated by vitamin D3 and insulin in the porcine ovarian follicles.

Dual-enhancement and dual-tag design for SERS-based sandwich immunoassays: evaluation of a metal-metal effect in 3D architecture.

The design of a sandwich-type SERS immunoassay (surface-enhanced Raman spectroscopy) is demonstrated operating in dual surface enhancement and dual-tag paradigm. The capture and detection antibodies are linked to two SERS-active substrates and form together the three-dimensional (3D) structure after specific binding to interleukin 6. A variety of metal combinations is tested (Au-Ag, Au-Au, and Ag-Ag), but an enhanced electromagnetic field is generated only due to coupling of Ag and Au nanoparticles with an Au hexagonal nanoarray. The amplified in that way Raman signals improve the limit of detection over 3 times in comparison to the assay with only one SERS-active substrate. It is also shown that the proper readout of the true-positive signal can be achieved in assays with two Raman tags, and this approach also improves LOD. For the optimal combination of the metal-metal junction and Raman tags, a linear relationship between the Raman signal and the concentration of IL-6 is obtained in the range 0-1000 pg⋅mL-1with LOD of 25.2 pg mL-1and RSD < 10%. The presented proof-of-concept of the SERS immunoassay with the dual-enhancement and dual-tag opens additional opportunities for engineering reliable SERS biosensing.

Molecular profiling of the intestinal mucosa and immune cells of the colon by multi-parametric histological techniques.

The impact of the post-mortem interval (PMI) on the optical molecular characteristics of the colonic mucosa and the gut-associated lymphoid tissue (GALT) were examined by multi-parametric measurements techniques. Inflammatory cells were identified by immunohistochemical staining. Molecular parameters were estimated using the Raman spectroscopy (RS) and Fourier Transform Infrared (FTIR) spectroscopic imaging. The 3D refractive index (3D-RI) distributions of samples were determined using the digital holographic tomography. The distribution of immune cells between post-mortem (PM) and normal controls did show significant differences for CD4 (P = 0.0016) or CD8 (P < 0.0001), whose expression level was decreased in PM cases. No association was found between individual PMI values and inflammatory cell distribution. However, there was a tendency for a negative correlation between CD4+ cells and PMI (r = - 0.542, P = 0.032). The alterations ongoing in post-mortem tissue may suggest that PMI has a suppressive effect on the effector properties of the cell-mediated immunity. Moreover, it was confirmed that spectroscopic and digital holotomographic histology are also a useful technique for characterization of the differences in inflammation of varying intensity and in GALT imaging in a solid tissue. Anatomical location of immune cells and methods of tissue fixation determine the molecular and optical parameters of the examined cases.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, 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 highly 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 interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics 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 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different 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.

Fourier transform infrared spectroscopic signature of blood plasma in the progression of breast cancer with simultaneous metastasis to lungs.

Despite advanced diagnostic techniques used for detecting cancer, this disease still remains a leading cause of death in the developed world. What is more, the greatest danger for patients is not related with growing of tumor but rather with metastasis of cancer cells to the distant organs. In this study, Fourier transform infrared (FTIR) spectroscopy was used to track chemical changes in blood plasma to find spectral markers of metastatic breast cancer during the disease progression. Plasma samples were taken 1-5 weeks after orthotropic inoculation of 4T1 metastatic breast cancer cells to mice. The earliest changes detected by FTIR spectroscopy in plasma were correlated with unsaturation of phospholipids and secondary structures of proteins that appeared 2 and 3 weeks, respectively, after 4T1 cells inoculation (micrometastatic phase). Significant alternations in the content and structure of lipids and carbohydrates were identified in plasma at the later stages (macrometastatic phase). When large primary tumors in breast and macrometastases in lung were developed, all bands in FTIR spectra significantly differed from those at earlier phases of the cancer progression. In conclusion, we showed that each phase of the breast cancer progression and its pulmonary metastasis can be characterized by a specific panel of spectral markers.

ImmunoSERS microscopy for the detection of smooth muscle cells in atherosclerotic plaques.

We investigated the suitability of immuno-SERS (iSERS) microscopy for imaging of smooth muscle cells (SMCs) in atherosclerotic plaques. Localization of SMCs is achieved by using SERS-labelled antibodies direct against alpha-smooth muscle actin (SMA). The staining quality of the false-colour iSERS images obtained by confocal Raman microscopy with point mapping is compared with wide-field immunofluorescence images. Both direct (labelled primary antibody) and indirect iSERS staining (unlabelled primary and labelled secondary antibody) techniques were employed. Direct iSERS staining yields results comparable to indirect IF staining, demonstrating the suitability of iSERS in research on atherosclerosis and paving the way for future multiplexed imaging experiments.

Raman Optical Activity and Raman spectroscopy of carbohydrates in solution.

This comprehensive study on selected 14 carbohydrates in water solution is an extension of previously published one focused only on solid state analysis. Here, Raman spectroscopy was used as a dedicated method for analysis of carbohydrates in solution, both using a normal effect (RS) and its chiral analogue: Raman Optical Activity spectroscopy (ROA). The compounds were selected as biologically important and representative of all groups: monosaccharides, disaccharides, trisaccharides, cyclodextrines and polysaccharides. RS and ROA spectra are presented together with an expanded discussion on various structures and conformations of studied carbohydrates in the solution taking into account particular regions, i.e. (1) low wavenumber region (250-600 cm-1), (2) anomeric region (600-950 cm-1), (3) fingerprint region (950-1200 cm-1) and (4) CH2and COH deformations region (1200-1500 cm-1). So, the following information can be obtained about: (1) the absolute configuration of the anomeric centre; (2) the configuration of the anomeric centre and the orientation of the anomeric hydroxyl group; (3) the ring structures and the relative orientation of substituents and (4) the conformation of the exocyclic CH2OH (4), respectively. Raman spectroscopy and Raman Optical Activity were shown as unique tools to study complex structures of carbohydrates.

Diversity among endothelial cell lines revealed by Raman and Fourier-transform infrared spectroscopic imaging.

Growing interest in the role of endothelium under physiological and pathological conditions has led to an increasing demand for its representative in vitro models especially suitable for drug tests and medical diagnostics. There are several endothelial cell lines commercially available whose biochemistry, and hence response to various stimuli, can be different. Recently, two vibrational techniques, Raman and Fourier-transform infrared microscopy, have been found to be potent tools for studying the biochemical composition of a single cell in an easy, rapid and label-free way. However, depending on the applied technique, the results may exhibit some divergence due to different selection rules as well as distinct experimental conditions. This paper presents the methodology of examination and characterization of three popular human endothelial cell lines: HAoEC (primary cells), HMEC-1 and EA.hy926 (immortalized cells). Based on high lateral resolution Raman imaging together with standard and high magnification Fourier-transform infrared measurements, the differences in spectral information and the distribution of biomolecules are presented and discussed.

FT-IR Spectroscopic Imaging of Endothelial Cells Response to Tumor Necrosis Factor-α: To Follow Markers of Inflammation Using Standard and High-Magnification Resolution.

Two endothelial cell lines were selected as models to investigate an effect of incubation with cytokine tumor necrosis factor type α (TNF-α) using Fourier transform infrared (FT-IR) imaging spectroscopy. Both cell lines are often used in laboratories and are typical lung vascular endothelial cells (HMLVEC) derived from the fusion of umbilical vein endothelial cells with lung adenocarcinoma cells (EA.hy926). This study was focused on alteration of spectral changes accompanying inflammation at the cellular level by applying two resolution systems of FT-IR microscopy. The standard approach, with a pixel size of ca. 5.5 µm2, determined the inflammatory state of the whole cell, while a high-magnification resolution (pixel size of ca. 1.1 µm2) provided information at the subcellular level. Importantly, the analysis of IR spectra recorded with different modes produced similar results overall and yielded unambiguous classification of inflamed cells. Generally, the most significant changes in the cells under the influence of TNF-α are related with lipids-their composition and concentration; however, segregation of cells into subcellular compartments provided an additional insight into proteins and nucleic acids related events. The observed spectral alterations are specific for the type of endothelial cell line.

A possible Fourier transform infrared-based plasma fingerprint of angiotensin-converting enzyme inhibitor-induced reversal of endothelial dysfunction in diabetic mice.

Angiotensin-converting enzyme inhibitors (ACE-I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)-based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased ß-sheet formation. Spectral markers at 3010, 1520/1238 cm-1 , representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE-I.

Raman and infrared spectroscopy of carbohydrates: A review.

Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (d-(-)-ribose, 2-deoxy-d-ribose, l-(-)-arabinose, d-(+)-xylose, d-(+)-glucose, d-(+)-galactose and d-(-)-fructose) and disaccharides (d-(+)-sucrose, d-(+)-maltose and d-(+)-lactose), and then more complex ones, i.e. trisaccharides (d-(+)-raffinose) and polysaccharides (amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050cm-1), IV (3050-2800cm-1) and II (1200-800cm-1) assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively, and region III (1500-1200cm-1) and I (800-100cm-1) dominated by deformational modes of the CH/CH2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure.

Raman microscopy as a novel tool to detect endothelial dysfunction.

Raman microscopy, a label-free method with high spatial resolution, shows growing potential in various fields of medical diagnostics. Several proof-of-concept studies related to the application of Raman microscopy to detect endothelial dysfunction are summarized in this work. Both ex vivo measurements of the tissues in the murine models of endothelial pathologies, as well as in vitro investigations of the cell cultures in the context of cellular transport, drug action and inflammation processes are discussed. The future directions in application of Raman spectroscopy-based methods in such studies are also described.