Spectrofluorometric quantification of neutral and polar lipids in zooplankton using Nile red

This study defines the conditions (wavelength selection and sensitivity) for spectrofluorometric quantification of neutral and polar lipids using Nile red (NR). The fluorescence intensity of the probe with standard lipids was examined in relation to lipid concentration and composition. Standards used included commercial triolein (TO) and phosphatidylcholine (PC), triacylglycerols (TG) and phospholipids (PL) from the krill Euphausia superba and Meganyctiphanes norvegica, and wax esters (WE) and PL from the copepods Paraeuchaeta antarctica and Drepanopus pectinatus. At any wavelength tested, polar and neutral lipids could be detected and fluorescence overlapped resulting in an interference of 10% to 20%. This interference between polar and neutral lipids was quantified for the standardisation. Wavelength selection for maximum emission with neutral and polar lipid classes achieved maximal sensitivity. Relationships between fluorescence intensity and lipid concentration were shown to be linear and specific to the lipid used. Large errors occurred when calibrations were performed with commercial lipids or with lipids obtained from other plankton species. Therefore, only the use of specific lipids as standards provided simultaneous and absolute quantification of neutral and polar lipids (relative errors were lower than 5%). The presence of PL in the sample (a ratio as low as 1:20) improved fluorescence of NR with TG by enhancing the solubility of the lipid microemulsion in water.