triolein and nile-red

Nile red (NR) is a popular fluorescent indicator to visualize lipid bodies in intact cells and has been extensively utilized to monitor triglyceride accumulation in microalgae. Typically, addition of NR to algae results in a rapid fluorescence enhancement followed by fluorescence quenching. NR fluorescence rise can be resolved into two kinetic phases: a fast phase (P₁, sec), monitored at 525 nm/630 nm, followed by a slower phase (P₂, min), monitored at 488 nm/575 nm. Studies with isolated plasma membrane (PM) and lipid globule (LG) preparations, suggest that P₁ and P₂ represent entry to the PM and transfer to LG, respectively. High NaCl slows down the interactions of NR with algae and with lipid globules. The onset of NR fluorescence quenching varies in different algae species between 5 min to 1h, and is observed in intact cells and in isolated LG. NR fluorescence quenching depends on NR concentration and is almost eliminated at low NR/cell ratios, indicating that it results from self-interactions of LG-associated dye. Glycerol has a dual effect on NR fluorescence: it eliminates kinetic anomalies resulting from limited solubility and self-interactions, but it also quenches NR fluorescence. NR fluorescence quenching by glycerol, as well as NR fluorescence enhancement by iodide anions, was observed only at high NR/LG ratios. These findings suggest that lipid-associated NR is more exposed to hydrophilic quenchers at high than at low NR concentrations. The results emphasize the importance of defining the optimal time window and NR concentrations for monitoring lipid accumulation in microalgae by NR fluorescence and clarify the origin of spectral anomalies resulting from self-interactions of dye molecules.

Topics: Cell Membrane; Fluorescent Dyes; Glycerol; Kinetics; Liposomes; Microalgae; Oxazines; Phosphatidylcholines; Spectrometry, Fluorescence; Triolein

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source. Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve. Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields. The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).

Topics: Calibration; Fluorescent Dyes; Lipids; Mediterranean Sea; Microalgae; Oxazines; Reference Standards; Sensitivity and Specificity; Spectrometry, Fluorescence; Staining and Labeling; Triolein