Open Access

Show Abstract ▼

Abstract: Cardiovascular diseases are still among the leading causes of mortality and morbidity worldwide. The build-up of fatty plaques in the arteries, leading to atherosclerosis, is the most common cause of cardiovascular diseases. The central player in atherosclerotic plaque formation is the foam cell. Foam cells are formed when monocytes infiltrate from the blood stream into the sub-endothelial space, differentiating into macrophages. With the subsequent uptake and storage of lipoprotein, especially low-density lipoprotein (LDL), they change their phenotype to lipid laden cells. Lowering circulating LDL levels, or initiating cholesterol efflux/reverse cholesterol transport in foam cells, is one of the current clinical therapies. Prescription of the pleiotropic drugs, statins, is the most successful therapy for the treatment and prevention of atherosclerosis. In this study, we used a foam cell model from the macrophage cell line, RAW 246.7, and applied the label-free Fourier Transform Infrared Spectroscopy (FTIR) method, i.e. synchrotron-based microFTIR spectroscopy, to study the lipid efflux process initiated by statins in a dose and time dependent manner. We used glass coverslips as substrates for IR analysis. The optical images (visible and fluorescent light) clearly identify the localization and lipid distribution within the foam cells, and the associated changes before and after culturing them with atorvastatin at concentrations of 0.6, 6 and 60 μg mL−1, for a culture duration between 24 to 72 hours. MicroFTIR spectroscopic spectra uniquely displayed the reduction of lipid content, with higher lipid efflux observed at higher doses of, and longer incubation time with, atorvastatin. Principal Component Analysis (PCA) and t-distributed Stochastic Neighbor Embedding (t-SNE) analysis demonstrated defined cluster separation at both lipid (3000–2800 cm−1) and fingerprint (1800–1350 cm−1) regions, with more profound discrimination for the atorvastatin dose treatment than time treatment. The data indicate that combining synchrotron-based microFTIR spectroscopy and using glass substrates for foam cells can offer an alternative tool in atherosclerosis investigation at a molecular level, and through cell morphology.

Open Access

Show Abstract ▼

Abstract: Photoacoustic spectroscopy (PAS) measures the photon absorption spectrum of a sample through detection of the acoustic wave generated by the photothermal effect as one modulates the intensity of the incident radiation at each wavelength. We have recently demonstrated the implementation of PAS in a microscopy configuration with mid-infrared radiation (microPAS). In the present work, we describe the performance of microPAS using synchrotron radiation (SR) in diffraction-limited spectromicroscopy and imaging experiments. Spectra were obtained for polystyrene beads, polypropylene fibres, and single fibres of human hair. SR produced microPAS spectra of much higher intensity as compared with those obtained using conventional mid- and near-infrared sources. For hair samples, the penetration depth of mid-infrared light, even with bright SR, is significantly shorter than the probed sample thickness at very low modulation frequencies resulting in saturated PAS spectra. In contrast, microPAS spectra of polymer beads were in general of much better quality than those obtained with conventional sources. We also demonstrated the capability to collect line profiles and line spectra at diffraction limited spatial resolution. The microPAS spectra of beads appear free from appreciable bandshape distortions arising from the real part of the refractive index of the sample. This observation confirms microPAS as an absorption-only technique and establishes it as a valuable new tool in the microspectroscopic analysis of particulates and of samples with a complex topography.

Show Abstract ▼

Abstract: Isolated and monolayer expanded chondrocytes are not the ideal cell form to produce cartilage matrix. In articular cartilage, each chondrocyte is surrounded by a 2-4 µm thick collagen VI-rich pericellular matrix (PCM) forming a chondron. Freshly extracted chondrons form a more cartilage-like extracellular matrix (ECM) than chondrocytes and their surrounding PCM is thought to maintain chondrocyte phenotype. To regenerate articular cartilage, preserving and/or regenerating a functional PCM is essential. In this study, a highly biomimic hyaluronic acid (HA) hydrogel was used as a 3-dimensional system to culture freshly isolated bovine chondrons (with an intact PCM) and chondrocytes (without a PCM) for up to 21 days. We assessed the HA hydrogels capacity to maintain and potentially re-generate PCM formation by both biochemical and immunological analyses for key components of the PCM. For the first time, Synchrotron based Fourier Transform Infrared microspectroscopy was utilised to reveal the dynamic process of PCM re-generation. At day 1, highly specific collagen VI staining was visible within chondron containing HA hydrogels. By contrast, collagen VI was absent at day 1 but punctate, focal staining increased during the culture period of chondrocyte containing HA hydrogels. Chondron containing HA hydrogels produced more collagen II and GAG than the chondrocyte containing HA hydrogels. Principal component analysis (PCA) of spectra in fingerprint regions on the chondrocyte-containing constructs at day 7, 14 and 21 culturing showed clear spectral differences. The clusters of day 14 and day 21 samples were closer to chondron samples, whilst day 7 samples were closer to chondrocytes. PCA scores in the lipid region revealed no major differences between chondrocytes and chondron samples, but differences between samples at day 7, day 14 and day 21. These data would indicate that SR-FTIR micro-spectroscopy can help to better understand the PCMs formation and maturation in tissue engineered models, which involves subtle changes in collagen and aggrecan.

Show Abstract ▼

Abstract: We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210BCR/ABL (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800–850 cm−1 frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia.

Show Abstract ▼

Abstract: We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210BCR-ABL drug-sensitive wild-type BCR-ABL or the V299L or T315I p210BCR-ABL drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 ?m2 in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of >95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.