title: Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy.
abstract: Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation.
authors: Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
authors: Daniel
authors: D
authors: Jaeger
authors: Biomolecular Photonics, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany.
authors: Christian
authors: C
authors: Pilger
authors: Biomolecular Photonics, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany.
authors: Henning
authors: H
authors: Hachmeister
authors: Biomolecular Photonics, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany.
authors: Elina
authors: E
authors: Oberländer
authors: Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
authors: Robin
authors: R
authors: Wördenweber
authors: Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
authors: Julian
authors: J
authors: Wichmann
authors: Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
authors: Jan H
authors: JH
authors: Mussgnug
authors: Biomolecular Photonics, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany.
authors: Thomas
authors: T
authors: Huser
authors: Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
authors: Olaf
authors: O
authors: Kruse
mesh_terms: D001709:Biotechnology; D002734:Chlorophyll / Q000737:chemistry; D000460:Chlorophyta; D005227:Fatty Acids / Q000737:chemistry; D066292:Lipid Droplets; D008055:Lipids / Q000737:chemistry; D058086:Microalgae / Q000378:metabolism*; D008853:Microscopy; D010788:Photosynthesis; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013818:Thermogravimetry
chemical_list: D005227:Fatty Acids; D008055:Lipids; D002734:Chlorophyll