Rapid Detection of Benzo[a]pyrene in Extra Virgin Olive Oil Using Fluorescence Spectroscopy.

Extra virgin olive oil (EVOO) should be naturally free of polycyclic aromatic hydrocarbon (PAH) contamination. PAHs are carcinogenic and toxic, and may cause human health and safety problems. This work aims to detect benzo[a]pyrene residues in EVOO using an easily adaptive optical methodology. This approach, which is based on fluorescence spectroscopy, does not require any sample pretreatment or prior extraction of PAH content from the sample, and is reported for the first time herein. The detection of benzo[a]pyrene even at low concentrations in extra virgin olive oil samples demonstrates fluorescence spectroscopy's capability to ensure food safety.

Comparative Evaluation of Different Targeted and Untargeted Analytical Approaches to Assess Greek Extra Virgin Olive Oil Quality and Authentication.

Extra virgin olive oil (EVOO) is a key component of the Mediterranean diet, with several health benefits derived from its consumption. Moreover, due to its eminent market position, EVOO has been thoroughly studied over the last several years, aiming at its authentication, but also to reveal the chemical profile inherent to its beneficial properties. In the present work, a comparative study was conducted to assess Greek EVOOs' quality and authentication utilizing different analytical approaches, both targeted and untargeted. 173 monovarietal EVOOs from three emblematic Greek cultivars (Koroneiki, Kolovi and Adramytiani), obtained during the harvesting years of 2018-2020, were analyzed and quantified as per their fatty acids methyl esters (FAMEs) composition via the official method (EEC) No 2568/91, as well as their bioactive content through liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) methodology. In addition to FAMEs analysis, EVOO samples were also analyzed via HRMS-untargeted metabolomics and optical spectroscopy techniques (visible absorption, fluorescence and Raman). The data retrieved from all applied techniques were analyzed with Machine Learning methods for the authentication of the EVOOs' variety. The models' predictive performance was calculated through test samples, while for further evaluation 30 commercially available EVOO samples were also examined in terms of variety. To the best of our knowledge, this is the first study where different techniques from the fields of standard analysis, spectrometry and optical spectroscopy are applied to the same EVOO samples, providing strong insight into EVOOs chemical profile and a comparative evaluation through the different platforms.

Optical spectroscopy methods combined with multivariate statistical analysis for the classification of Cretan thyme, multi-floral and honeydew honey.

BACKGROUND: The botanical origin of honey attracts both commercial and research interest. Consumers' preferences and medicinal uses of particular honey types drive the demand for the determination of their authenticity with regard to their botanical origin. This study presents the discrimination of thyme, multi-floral. and honeydew honeys by Fourier-transform infrared (FTIR) and ultraviolet (UV) absorption spectroscopy combined with multivariate statistical analysis. UV absorption spectroscopy was applied without any dilution of the sample using a custom-made cuvette. FTIR and UV absorption spectroscopic data were processed by means of the orthogonal partial least squares discriminant analysis. RESULTS: The optimal classification of floral and honeydew honeys was accomplished with UV spectroscopy with a successful estimation of 92.65% for floral honey and 91.30% for honeydew honey. The discrimination of thyme versus the multi-floral honey was best achieved with FTIR, with a correct classification of 95.56% and 100% for multi-floral and thyme honey respectively. Furthermore, our findings revealed the region of 2400-4000 cm-1 of the FTIR spectra as the most significant for this discrimination. CONCLUSION: This work demonstrates that optical spectroscopic techniques in combination with multivariate statistical analysis can be a rapid, low-cost, easy-to-use approach for the determination of the botanical origin of honey without sample pretreatment. © 2021 Society of Chemical Industry.

Application of Ultraviolet-Visible Absorption Spectroscopy with Machine Learning Techniques for the Classification of Cretan Wines.

The present study was aimed at the identification, differentiation and characterization of red and white Cretan wines, which are described with Protected Geographical Indication (PGI), using ultraviolet-visible absorption spectroscopy. Specifically, the grape variety, the wine aging process and the role of barrel/container type were investigated. The combination of spectroscopic results with machine learning-based modelling demonstrated the use of absorption spectroscopy as a facile and low-cost technique in wine analysis. In this study, a clear discrimination among grape varieties was revealed. Moreover, a grouping of samples according to aging period and container type of maturation was accomplished, for the first time.

Verifying the Geographical Origin and Authenticity of Greek Olive Oils by Means of Optical Spectroscopy and Multivariate Analysis.

Olive oil samples from three different Greek regions (Crete, Peloponnese and Lesvos) were examined by optical spectroscopy in a wide spectral region from ultraviolet to near infrared using absorption, fluorescence and Raman spectroscopies. With the aid of machine learning methods, such as multivariate partial least squares discriminant analysis, a clear classification of samples originating from the different Greek geographical regions was revealed. Moreover, samples produced in different subareas of Crete and Peloponnese were also well discriminated. Furthermore, mixtures of olive oils from different geographical origins were studied employing partial least squares as a tool to establish a model between the actual and predicted compositions of the mixtures. The results demonstrated that optical spectroscopy combined with multivariate statistical analysis can be used as an emerging innovative alternative to the classical analytical methods for the identification of the origin and authenticity of olive oils.

Spatial distribution and function of sterol regulatory element-binding protein 1a and 2 homo- and heterodimers by in vivo two-photon imaging and spectroscopy fluorescence resonance energy transfer.

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

Nonlinear optical microscopy of articular cartilage.

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy.

The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.

Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level.

We have employed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of green fluorescent protein (GFP) mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Utilizing the intrinsic optical sectioning capabilities of TPIS-FRET, we have identified protein-protein interactions within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.

Multiphoton microscopy relies on nonlinear light-matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue "intravital" imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730-880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. Endogenous cellular signals are consistent with TPEF spectra of cofactors NAD(P)H and FAD at all excitation wavelengths. The reflected SHG intensity follows a quadratic dependence on the excitation power, decays exponentially with depth, and exhibits a spectral dependence in accordance with previous theoretical studies. The use of SHG and TPEF in combination provides complementary information that allows noninvasive, spatially localized in vivo characterization of cell-ECM interactions in unstained thick tissues.

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence.

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.