Supplementary MaterialsSupplementary Information srep35340-s1. comprising triacylglycerols (TAGs)4. TAGs, stored as lipid droplets (LDs) within the cell, are of great interest for biodiesel production4,5. Proteome studies of isolated LDs of the model green alga have revealed a diverse set of proteins, of which more than 30 were involved in lipid metabolism6,7. The most abundant protein was termed (MLDP) and was shown to act as a scaffold stabilizing LD size, and furthermore proposed to recruit other proteins to the LDs, in particular tubulin8. Lipid quantification is routinely conducted by solvent extraction (for example, Folch extraction9), followed by fractionation and gravimetrical analysis. Subsequent HPLC or LY2140023 kinase inhibitor GC-MS measurements can be performed to elucidate the fatty acid composition (fatty acid profile) of the isolated lipids. Most of the established lipid extraction and characterization protocols are laborious and require a substantial amount of biomass3. Alternatively, lipid-specific fluorophores such as Nile Red can be employed to quantify the lipid content by fluorescence detection10. A powerful approach is to combine fluorophore staining with flow cytometry to rapidly monitor lipid accumulation11, or LY2140023 kinase inhibitor with LY2140023 kinase inhibitor fluorescent activated cell sorting to separate high-lipid mutants from a collection of transformants12. A major disadvantage of fluorophore staining is that many microalgal species are enclosed by a rigid cell wall, which can act as a permeability hurdle for the dye10,13. Both, lipid composition and content, can be researched by Raman micro-spectroscopy, a label-free and non-destructive technique which depends on Raman scattering by probing the vibrational personal of substances14. Raman spectroscopy produces home elevators the molecule classes in the cell or mobile compartment, and may, for example, be utilized to derive the comparative amount of unsaturation of essential fatty acids inside a subcellular test15. Raman scattering can be the basis for CARS, by which the distribution of a single molecular bond inside a sample can be imaged16,17. In the context of lipid analysis, aliphatic C-H2 bonds can be probed, which LY2140023 kinase inhibitor are specifically 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 thus be assessed in a label-free manner, which is a promising, cell-wall independent alternative for FA3 lipid quantification in a broad range of species. Compared to spontaneous Raman imaging with typical pixel dwell times of several seconds18,19,20, pixel dwell times in CARS imaging are in the range of microseconds21 and thus significantly faster due to a 105 times higher CARS signal compared to spontaneous Raman scattering22. This enables the analysis of dynamic processes inside living cells without any cell preparation such as LY2140023 kinase inhibitor immobilization. Furthermore, the z-resolution in CARS microscopy is in the range of 650?nm compared to 2000?nm18 for Raman microscopy, hence 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. This blue-shift is caused by the energy transfer from an excited bond vibration to a scattered photon (Stokes scattering refers to the red-shift of the signal photon wavelength that occurs while the photon interacts with the molecular bond vibration and loses energy due to the excitation of the vibrational bond). CARS microscopy employs a four-wave-mixing process to be able to positively probe molecular relationship vibrations appealing inside a pump-probe system17. Right here, an inbound pump photon with rate of recurrence along with a Stokes photon with rate of recurrence coherently excite the resonant molecular bonds collectively, while another photon (within the unique case of Vehicles with rate of recurrence C169. The analyses, nevertheless, had been tied to a substantial overlap between chlorophyll two-photon thrilled fluorescence as well as the engine cars signs24. Subsequently, Vehicles microscopy was requested the diatom is stained from the lipophilic fluorophore readily.