Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy |
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Authors: | Lillie Cavonius Helen Fink Juris Kiskis Eva Albers Ingrid Undeland Annika Enejder |
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Affiliation: | Division of Life Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE–412 96 Gothenburg, Sweden |
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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 (r2 = 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.The accumulation of lipids in microalgae is currently a field of intense research: with their high photosynthetic efficiency and rapid growth rates, these organisms hold great potential both for sustainable production of biofuels (Chisti, 2007) and as a nutrition source (de Jesus Raposo et al., 2013). As in all living cells, lipids in microalgae are present in membranes, such as the plasma and organelle membranes. Some microalgae also accumulate lipids, mainly triacylglycerols, in intracellular droplets (De Martino et al., 2011; White et al., 2012). One such microalga is Phaeodactylum tricornutum, a unicellular photoautotrophic diatom and a well-studied model organism. It has a sequenced genome and is known to produce long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and small amounts of docosahexaenoic acid (DHA; Alonso et al., 1998). The long-chain n-3 PUFA-producing properties has made it particularly interesting within the areas of functional food and nutraceuticals. Under conditions of nitrogen starvation, P. tricornutum accumulates larger amounts of fatty acids, albeit of the more saturated nature (Yongmanitchai and Ward, 1991). However, in response to low irradiance, P. tricornutum has been reported to increase its content of PUFAs, especially EPA (Thompson et al., 1990). In order to optimize strain selection and algal cultivation conditions in relation to lipid accumulation/lipid profile, accurate tools to quantify total lipids as well as the degree of unsaturation are required.Solvent extractions followed by methylation, gas chromatography coupled to flame ionization detection, and gas chromatography-mass spectrometry (GC-MS) detection belong to the standard techniques used for algal lipid analysis. However, these are cumbersome and require relatively large amounts of solvents and sample. The resulting quantitative information on lipid amounts is related to total cell mass, which may introduce artifacts, as the cell mass is also influenced by other metabolic parameters. Furthermore, these bioanalytical techniques provide rough population averages without information on the intracellular location or distribution. As an alternative, microscopy techniques are increasingly being used, with the benefit that lipid droplets can be evaluated directly in single cells with high precision. Fluorescence microscopy is the most widespread technique, relying on lipid-specific fluorescent markers: Nile Red is commonly used, but its poor permeability through the cell walls causes difficulties, as does its nonspecific binding (Chen et al., 2009). Other fluorophores like BODIPY 505/515 also have been suggested for live-cell studies (Cooper et al., 2010; Wong and Franz, 2013). Still, invasive techniques are needed, requiring solvents to facilitate the transport of the labeling molecules into the cell, potentially inducing stress responses that may affect cell metabolism. Furthermore, there is little knowledge available on how the accumulation of lipophilic dyes in lipid droplets and the fluorescence emission are influenced by environmental conditions such as temperature, pH, and deposited light doses. It has also been shown that the fluorescence intensity emitted from the dyes cannot be related directly to the local lipid concentration, excluding quantitative measurements (De la Hoz Siegler et al., 2012). In algae/plant cell biology, the applicability of fluorescence microscopy is also limited due to the fact that algae/plant cells generate strong autofluorescence, potentially interfering with the lipid/fatty acid signals. In order to take algal lipid quantification one step further, microscopy techniques, not being dependent on exogenous fluorophores and allowing efficient separation of the lipid/fatty acid signal from the autofluorescence, are desirable.In label-free coherent anti-Stokes Raman scattering (CARS) microscopy, images are formed by probing intrinsic molecular vibrations through a nonlinear four-wave-mixing process (Cheng and Xie, 2004). Briefly, the frequency difference of two coherent near-infrared excitation beams (the pump beam at shorter wavelengths and the Stokes beam at longer wavelengths) are tuned to match the frequency of the target molecular vibration. As a result, resonant oscillators are coherently driven in the sample focal volume and an enhanced blue-shifted CARS signal is generated. Due to the nonlinear nature of the CARS process, emission is generated only in the high-intensity region of focused laser beams, allowing optical sectioning of the specimen. By tuning the frequency difference of the excitation beams to match the resonance frequency of carbon-hydrogen (C-H) vibrations, especially abundant in lipids, three-dimensional images of lipids can be recorded without any staining (Enejder et al., 2010). As the CARS emission scales with the square of the concentration of C-H bonds, quantitative data on intracellular amounts of lipids can be extracted (Cheng and Xie, 2004). However, cells with chromophores, such as algae and plant cells, generate exceptionally strong two-photon fluorescence, the spectral tails of which tend to bleed through the most efficient optical filters typically used for separation of the CARS signal. As an alternative approach, we have incorporated a time-correlated single-photon counting system (Enejder et al., 2010), allowing us to distinguish the long-decay fluorescence component from the instant CARS signal by time gating.The capability of conventional CARS imaging for microalgae was recently demonstrated with proof-of-principle data, showing that individual, larger lipid droplets can be resolved visually, in contrast to conventional Raman microscopy (He et al., 2012). In this study, we introduce CARS microscopy with time-gated detection, also enabling the identification of subpicogram lipid-rich regions in the vicinity of strongly autofluorescent chloroplasts. This is particularly important because cellular storage lipids in algae are primarily synthesized in the chloroplasts and then budded off from the envelope membranes as nascent lipid droplets (Fan et al., 2011). Hence, time-gated CARS microscopy paves the way for high-precision quantification of the complete intracellular distribution of lipid stores, including the emerging droplets within and adjacent to chloroplasts. We show the feasibility of the approach for quantitative lipid analysis of large populations of living P. tricornutum cultivated under three different growth conditions: a control (Ctrl) group, a light-limited (LL) group to increase the EPA level, and a nitrogen-starved (NS) group to increase total lipid accumulation. We studied the lipid metabolism in approximately 100 cells per category over time and report detailed visual information on the dynamics of lipid droplet formation throughout a life cycle from budding droplets to, in some cases, giant lipid stores. We show that biologically relevant quantitative and qualitative data on intracellular lipid stores can be extracted at a precision of less than 1 pg cell−1 despite adjacent chromophores. Data extracted from the CARS images are further compared with solvent extraction and quantification of fatty acids using GC-MS. We further illustrate the potential to assess whether the different growth conditions promote the synthesis of more PUFAs by detecting shifts in lipid fluidity and saturation per individual lipid droplet from CARSC-H vibration ratio images. |
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