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.
Highlights
Photosynthetic microalgae have recently gained significant attention as sustainable production hosts for a range of bio-products[1,2,3]
The z-resolution in coherent anti-Stokes Raman scattering (CARS) microscopy is in the range of 650 nm compared to 2000 nm[18] for Raman microscopy, the detailed lipid distribution can be imaged in 3D, especially in the case of small lipid droplets (LDs)
We present an approach to separate the CARS signal from the two-photon excited chlorophyll fluorescence utilizing a modulation scheme
Summary
Photosynthetic microalgae have recently gained significant attention as sustainable production hosts for a range of bio-products[1,2,3]. In the context of lipid analysis, aliphatic C-H2 bonds can be probed, which are enriched in the lipid fraction, in particular in LDs. Utilizing optical microscopy with CARS as a contrast mechanism, the distribution of lipids inside the cell can be assessed in a label-free manner, which is a promising, cell-wall independent alternative for lipid quantification in a broad range of species. The z-resolution in CARS microscopy is in the range of 650 nm compared to 2000 nm[18] for Raman microscopy, the detailed lipid distribution can be imaged in 3D, especially in the case of small LDs. CARS is based on anti-Stokes scattering, a process in which a blue-shift of the photon wavelength is detected. In order to separate the CARS signal from the undesired two-photon excited fluorescence of chlorophyll which spectrally overlaps with the CARS signal wavelengths, a very narrow band-pass filter that isolates the CARS wavelength of the C-H2 stretching mode and, a modulation technique can be applied[23]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
