Microalgal lipid bodies: Detection and comparative analysis using imaging flow cytometry, confocal laser scanning and Raman microscopy.

In this study, imaging flow cytometry (iFCM), confocal laser scanning microscopy (CLSM) and Raman microscopy were compared for their ability to detect lipid bodies in Phaeodactylum tricornutum (a diatom) and Nannochloropsis oculata. These two microalgae belonging to a different phylum were cultivated under standard growth conditions and analysed in the mid-exponential and early stationary growth phase. Lipids were detected using Nile red as a fluorescent dye. Spectral unmixing was applied in CLSM to separate the fluorescent signals from neutral lipids, polar lipids and pigments. CLSM was able to detect lipid bodies in a higher number of cells compared to iFCM or Raman microscopy. Pigments were interfering with the lipid body detection in iFCM while proteins were interfering in Raman microscopy. Nevertheless, the increase in lipid body area per cell from the mid-exponential phase to the early stationary growth phase follows a similar trend for both CLSM and iFCM. • Neutral lipid, polar lipid and pigment fluorescence were split by spectral unmixing. • CLSM detects lipid bodies in more cells compared to other techniques tested. • Similar increase of lipid body area per cell by CSLM and imaging flow cytometry • Lipid bodies smaller than 0.8 μm in radius were not detected by Raman microscopy. • Interference of proteins potentially hampers lipid detection in Raman microscopy. [ABSTRACT FROM AUTHOR]