Raman imaging highlights biochemical heterogeneity of human eosinophils versus human eosinophilic leukaemia cell line.

Eosinophils are acidophilic granulocytes that develop in the bone marrow. Although their population contributes only to approximately 1-6% of all leucocytes present in the human blood, they possess a wide range of specific functions. They play a key role in inflammation-regulating processes, when their numbers can increased to above 5 × 109 /l of peripheral blood. Their characteristic feature is the presence of granules containing eosinophil peroxidase (EPO), the release of which can trigger a cascade of events promoting oxidative stress, apoptosis or necrosis, leading finally to cell death. Raman spectroscopy is a powerful technique to detect EPO, which comprises a chromophore protoporphyrin IX. Another cell structure associated with inflammation processes are lipid bodies (lipid-rich organelles), also well recognized and imaged using high resolution confocal Raman spectroscopy. In this work, eosinophils isolated from the blood of a human donor were analysed versus their model, EoL-1 human eosinophilic leukaemia cell line, by Raman spectroscopic imaging. We showed that EPO was present only in primary cells and not found in the cell line. Eosinophils were activated using phorbol 12-myristate 13-acetate, which resulted in lipid bodies formation. An effect of cells stimulation was studied and compared for eosinophils and EoL-1.

Raman spectroscopic features of primary cardiac microvascular endothelial cells (CMECs) isolated from the murine heart.

Gaining knowledge on the biochemical profile of primary endothelial cells on a subcellular level can contribute to better understanding of cardiovascular disease. In this work, primary cardiac microvascular endothelial cells (CMECs) isolated from the mouse heart and murine H5V endothelial cell line were characterized with the use of a Raman imaging technique. Primary CMECs displayed a distinct Raman-based biochemical phenotype as compared with other cells isolated from the heart and were characterized by a low lipid content. In contrast to the murine H5V endothelial cell line, CMECs did not display lipid droplets (LDs) in the cytoplasm, while the former have many low-unsaturated LDs. In conclusion, Raman imaging is a fast and efficient tool to analyse single coronary endothelial cells in a non-invasive manner that can prove useful to characterize biochemical changes in a single isolated primary endothelial cell from a diseased heart.

Influence of polar and nonpolar carotenoids on structural and adhesive properties of model membranes.

Carotenoids, which are known primarily for their photoprotective and antioxidant properties, may also strongly influence the physical properties of membranes. The localization and orientation of these pigments in the lipid bilayer depends on their structure and is determined by their interactions with lipid molecules. This affects both phase behavior and the mechanical properties of membranes. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) allowed us to gain a direct insight into the differences between the interaction of the non-polar ß-carotene and polar zeaxanthin embedded into DPPC liposomes. DSC results showed that zeaxanthin, having polar ionone rings, interacts more strongly with the membrane lipids than ß-carotene. The decrease in molar heat capacity by a factor of 2 with a simultaneous broadening of the main phase transition (gel-to-liquid crystalline phase transition) as compared to the two other systems studied suggests some increased length of the coupled interactions between the polar xanthophyll and lipids. Long-distance interactions lead to the formation of larger clusters which may exhibit higher flexibility than small clusters when only short-distance interactions occur. AFM experiments show that adhesive forces are 2 and 10 times higher for DPPC membranes enriched in ß-carotene and zeaxanthin, respectively, than those observed for an untreated system. Temperature dependent measurements of adhesion revealed that subphases can be formed in the gel lamellar state of DPPC bilayers. The presence of the non-polar carotenoid enhanced the effect and even a bifurcation of the substates was detected within a temperature range of 30.0-32.5°C prior to pretransition. It is the first time when the presence of subphases has been demonstrated. This knowledge can be helpful in better understanding the functioning of carotenoids in biological membranes. AFM seem to be a very unique and sensitive method for detecting such fine changes in the lipid bilayers.

Atomic force microscopy studies of the adhesive properties of DPPC vesicles containing ß-carotene.

A role of carotenoids as modulators of physical properties of model and biological membranes has been already postulated. However, there is a lack of information on the influence of these pigments on interactions between the lipids which form such membranes. This paper applies atomic force microscopy (AFM) in to study the effects of ß-carotene on the adhesion properties of DPPC multilamellar liposomes. This allowed us to gain, for the first time, a direct insight into the interactions between the components in model systems on a molecular level. We observe that the adhesive forces in DPPC multilamellar liposomes containing 1mol% of ß-carotene decrease exponentially with increasing temperature, and that at about 37°C they diminish. In the case of pure liposomes the decline in adhesion is of a different nature and the adhesive forces disappear at 34°C. The adhesive forces are about 5 times higher at 31°C in the presence of ß-carotene than in its absence. However, measurements using differential scanning calorimetry (DSC) showed a shift of the lamellar-to-undulled-lamellar phase transition toward lower temperatures by about 0.8 ± 0.2°C in a system containing ß-carotene. The enthalpy changes (ΔH) of this transition are similar for both systems. For the main transition, gel-to-liquid crystalline, the peak is shifted by about 0.5 ± 0.1°C, and ΔH decreases by about 30% in liposomes treated with ß-carotene in comparison to pure liposomes. Our results suggest increased cooperation between liposome components in a system with enriched ß-carotene, which cause a change in phase transition temperatures. Moreover, these interactions are very sensitive to temperature.