title: Imaging of lipids in microalgae with coherent anti-stokes Raman scattering microscopy.
abstract: Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r(2) = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.
authors.affiliation: Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Lillie
authors.initials: L
authors.lastname: Cavonius
authors.affiliation: Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Helen
authors.initials: H
authors.lastname: Fink
authors.affiliation: Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Juris
authors.initials: J
authors.lastname: Kiskis
authors.affiliation: Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Albers
authors.affiliation: Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Undeland
authors.affiliation: Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden enejder@chalmers.se.
authors.forename: Annika
authors.initials: A
authors.lastname: Enejder
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D005227:Fatty Acids / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D021621:Imaging, Three-Dimensional; D042541:Intracellular Space / Q000378:metabolism; D066292:Lipid Droplets / Q000378:metabolism; D008055:Lipids / Q000032:analysis*; D008560:Membrane Fluidity; D058086:Microalgae / Q000254:growth & development / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013997:Time Factors
chemical_list: D005227:Fatty Acids; D008055:Lipids