Lipidome of Activated Hepatic Stellate Cells Figure 3. HSC activation results in a decrease in retinyl esters in HSC LDs. Freshly isolated HSCs were cultured and fixed after 2 h (day 0; quiescent state), and day 4 and day 7 (activated state). Confocal Raman microscpectroscopy on LD enriched regions was performed as described in 2 the Method section. Cluster image (20620 mm ) was constructed from Raman imaging data of the square area in the white light image. Each color represents a different cluster. The cluster averages show the average Raman spectra extracted from the black, pink, green and blue clusters displayed in the cluster image. * indicates (characteristic) RE peaks; # indicates characteristic acyl peak. doi:10.1371/journal.pone.0034945.g003 already on day 4, which was even further decreased in the activated cells at day 7 (Fig. 3). (Activated) HSCs contain a homogenous LD population In the initial period of the activation process both large perinuclear LDs and small dynamic LDs in cell extensions could be observed. To investigate whether the lipid composition of both LD types was similar, confocal Raman microspectroscopy was performed on perinuclear and peripheral LDs in HSCs at day 0, 4 and 7. As can be seen in the cluster images in Fig. 3 the Raman spectra of all LDs at day 0 contain a similar profile, with the same ratio between the peak at 1595 cm21 (retinyl ester) and 1440 cm21 (TAG species). Also at day 4 and day 7 all LDs within one cell showed a similar spectrum, although as described above, different from that in quiescent HSCs. (Fig. 3; lower panels). These results indicate that HSCs contain a homogenous LD population with respect to their neutral lipid composition, which is independent of their size, their state of activation and/or localization. To measure the metabolic activity of the LDs, we applied deuterated arachidonic acid (20:4-d8/AA-d8; 25 mM) to HSCs. Deuteration allowed the discrimination of exogenously adminis- tered fatty acids from endogenous lipids by a characteristic peak around 2200–2300 cm21 of the C-D bond in the Raman spectrum [16]. All LDs at day 7 display a similar localization of the characteristic deuterium signal and the retinyl signal (Fig. 4A). This indicates that AA-d8 was incorporated in all LDs, both perinuclear and in the cell extensions. To obtain independent evidence for a similar metabolic activity of all LDs within one cell, we added the fluorescently labelled fatty acid, Bodipy-C12, to activated HSCs. Within one cell all LDs had incorporated similar levels of fluorescent fatty acid (Fig. 4B), although we could observe some differences in uptake between different cells plated at the same time. HSC activation results in a decrease in retinyl esters and an induction of polyunsaturated triacylglycerol species Our results suggest the presence of highly dynamic and metabolically active LDs. Therefore, we considered the possibility that both the lipid class profile and the lipid species (i.e. fatty acid) profile within a lipid class changed during HSC activation. We analysed the neutral lipid species profile of quiescent and activated PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34945