Nanoscopy

Raman spectroscopy is an attractive tool in identification cells, especially when studying cellular processes, including lipid droplets (LDs) formation. In this work, confocal Raman imaging was used to study the formation of LDs in vitro in a single endothelial cell (EC) upon incubation with polyunsaturated fatty acids (PUFAs, 10 or 25 µM) including arachidonic acid (AA) and its deuterated analog (AA-d8), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Based on Raman spectra taken from a single EC, it was possible to investigate biochemical changes induced by addition of PUFA. In particular, content of lipids in LDs induced by PUFA as well as their unsaturation was identified by Raman spectroscopy by marker bands at 1660 cm−1 due to C=C stretching and ~3015 cm−1 due to the stretching mode of =C-H associated with C=C double bonds (except for a deuterated form where these bands are shifted respectively). To establish if exogenous fatty acid was taken up by the cell and stored in LDs, a deuterium labelled PUFA was used. AA-d8 shows distinct characteristic bands around 2200–2300 cm−1 assigned to =CD stretching modes. We demonstrated that AA and EPA was taken up by endothelial cells into their newly formed LDs, while DHA was not, even though LDs were formed. Furthermore, using AFM we demonstrated that the presence of LDs in the endothelium affected endothelial stiffness which could have pathophysiological significance. In summary, the results suggest that formation of LDs in endothelium involve exogenous and endogenous PUFA, and their relative contribution seem distinct for AA, EPA and DHA.