Lipidomic profiles of tissue extracts have contributed substantially to elucidating the plant lipidome and its changes in association with physiological conditions. However, the spatial organization of lipids in tissues, cells and subcellular compartments is mostly lost as a result of chemical-based extraction of plant tissues. Characterization of the location of lipids in plant tissues and cells is at least as important to determining the complexity of the lipidome and its quantification. The cellular distribution of lipids can be partially resolved using optical imaging techniques such as confocal or electron microscopy with selected fluorescent dyes, antibodies and chemical modifications (Eggeling et al., 2009; Wessels et al., 2010), but visualizing the detailed composition of individual molecular species is limited using conventional microscopy approaches. The emerging field of mass spectrometry imaging (MSI) captures the spatial distribution of lipids in situ, with detailed compositional information at the tissue, cellular, and, with some instrument modifications, subcellular levels. Several spectrometers are available for MSI, which vary in terms of ionization, detection sensitivity, spatial and spectral resolution, and the type of information acquired. Some instruments require advanced sample preparation but overall enable a diversified approach towards analyzing the lipidome by MSI. Although there have been several comprehensive reviews of MSI (Chaurand et al., 2005; Dill et al., 2011; Harris et al., 2011; Lee et al., 2012), here we discuss methods for visualizing plant lipids at the cellular level – focusing on matrix-assisted laser desorption/ionization MS (MALDI-MS) and desorption electrospray ionization (DESI). New techniques allow the analysis of lipids at the subcellular level, such as direct organelle MS (DOMS) and live single-cell MS. Collectively, these techniques provide plant scientists with the opportunity to obtain a breadth of chemical composition information within a biologically relevant spatial context (see Table 1 for summary of methods discussed). Some alternative MS approaches for analyzing lipids in plant cells are also mentioned below, including secondary ion MS (SIMS) and ion mobility MS (IMMS).
Matrix-assisted laser desorption/ionization
Matrix-assisted laser desorption/ionization MS is a versatile imaging platform that is particular amenable for sampling biological tissues. Although MALDI has traditionally been utilized to analyze larger biomolecules such as proteins and peptides, it is increasingly being applied to characterizing lipid extracts and visualizing lipids in situ. Tissues are imaged by preparing thin sections that can be coated with a suitable matrix that limits sample damage and promotes ionization. The choice of matrix (e.g. dihydroxybenzoic acid, cinnamic acid, etc.) and the method by which it is applied to the sample (e.g. sublimation) are important considerations as they may significantly affect ionization efficiency and the resulting compositional analysis. Samples coated with matrix are then ionized, as described in detail previously (Zenobi and Knochenmuss, 1998). Briefly, a laser ablates a region of tissue, and both matrix- and tissue-derived ions are directed towards a mass analyzer. The laser is rastered over the tissue sample, and mass spectra are collected at each location, producing a chemical map of the plant material. A resolution of 10–50 μm is typically used for laser-based imaging, with some reports of submicron MALDI imaging (Guenther et al., 2010). As ionization typically occurs under vacuum, sample preservation and preparation, in addition to the compatibility of the ionization matrix, are key factors for comprehensive analysis and compositional integrity. Quantification of lipid species in tissues and sample extracts by MALDI-MS is limited due to suppression of certain lipid classes [i.e. PC generally suppresses other phospholipids and TAGs (Emerson et al., 2010)]. Validation using conventional shotgun lipidomics, as well the inclusion of internal standards within or in addition to the matrix, may resolve suppression-influenced quantification.
Several studies have used MALDI-TOF MS to profile plant lipid extracts as an alternative to separation-based MS methods. These MALDI-TOF MS data also support standardization of the platform for future in situ analysis. An in-depth study on PC species extracted from soybean (Glycine max) and potato tubers (Solanum tuberosum) found that the individual isomeric PC species could be resolved by enzymatic digestion of PC species and analysis by MALDI-TOF in full-scan mode, and post-source decay of fragments generated during their flight in the mass spectrometer (Zabrouskov et al., 2001). Similar methodology was used successfully for analysis of the glycerophospholipid and glycolipid species of the plant-like green alga Chlamydomonas reinhardtii (Vieler et al., 2007). Lipid-soluble betacyanin pigments, utilized for food colorants and as antioxidants, were extracted from Amaranthus tricolor seedlings, Gomphrena globosa flowers and Hylocereus polyrhizus fruits, and structurally characterized by MALDI-quadrupole ion trap-TOF MS (Cai et al., 2006). Similar characterization was performed for chlorophyll and its derivatives extracted from spinach (Spinacia oleracea L.) leaves using MALDI-TOF MS (Suzuki et al., 2009). The TAG and fatty acid compositions of total lipid extracts from olive (Olea europaea) and pomegranate (Punica granatum) seeds have been standardized using MALDI-TOF as an alternative to fatty acid analysis by gas chromatography for quality assurance in industrial production of seed oils (Wiesman and Chapagain, 2010).
In terms of imaging lipids in situ, most analysis by MALDI-MS to date has focused on mammalian tissues in the context of genetic disorders of lipid metabolism (Murphy et al., 2009; Fuchs et al., 2010). Recently, however, MALDI-MS has been used for in situ analysis of plant lipids and several other types of metabolites (Kaspar et al., 2011). Surface lipids were imaged directly in Arabidopsis floral and leaf tissues by MALDI-MS (Cha et al., 2008; Jun et al., 2010; Vrkoslav et al., 2010). Elsewhere, MALDI-MS using colloidal graphite as a matrix for ionization was used to image the free fatty acids in strawberry seeds (Fragaria × ananassa) and apple tissues (Malus domestica) (Zhang et al., 2007). MSI in rice grains (Oryza sativa) identified PC molecular species (Zaima et al., 2010).
Infrared-laser desorption/ionization oTOF MS has been used to directly profile metabolite changes in defense responses in tobacco leaves (Nicotiana tabacum) against the oomycete Phytophthora nicotianae (Ibanez et al., 2010). These time-dependent profiling experiments identified a group of peroxidized oxylipins, which can act as stress signaling molecules, and their precursor γ-linolenic acid, in response to pathogen infection. This technique was also used to generate lipid profiles (i.e. TAGs, fatty acids, PCs, etc.) directly from pieces of green olive (Olea europaea), sesame seed (Sesamum indicum), sunflower seed (Helianthus annuus L.), white coconut flesh (Cocos nucifera L.), strawberry seeds (Fragaria x ananassa) , and a red rose leaf (Rosa sp. cv El toro), which demonstrates the diversity of plant tissues that can be quickly profiled (Dreisewerd et al., 2007). Atmospheric pressure infrared MALDI-MS was used to obtain profiles from various plant organs, including TAGs within sections of almond seeds (Prunus amygdalus) (Li et al., 2007). Laser ablation ESI (LAESI) was used to profile the 2D distribution (with approximately 350 μm lateral resolution and approximately 50 μm depth resolution) of metabolites within the polyketide kaempferol biosynthesis pathway in leaf tissue of the variegated zebra plant (Aphelandra squarrosa) (Nemes et al., 2008). Similar imaging analysis was performed to reconstruct 3D metabolite profiles of the leaves of A. squarrosa and Spathiphyllum lynise (peace lily), including the kaempferol pathway and chlorophyll pigments (Nemes et al., 2009). Localizing lipid metabolites directly in plant tissues provides a new basis for lipidomics at the cellular level, and will provide important spatial information for studies of lipid metabolism and function.
Desorption electrospray ionization (DESI)
Desorption electrospray ionization is an ambient ionization technique that combines features of electrospray and desorption ionization for direct tissue analysis (Harris et al., 2011). DESI ionizes molecules by generating pneumatically assisted charged droplets directed towards the tissue surface that, upon collision, give rise to secondary droplets from compounds on the tissue surface that can be analyzed by high-resolution MS (Takáts et al., 2004). DESI-MS is an attractive technique for MSI applications as it requires little to no sample preparation (no matrix required) compared with MALDI-MS, and samples are not introduced into high-vacuum conditions (Harris et al., 2011). A major limitation of DESI is that its mode of ionization results in poor spatial resolution: of the order of 100–250 μm, which is generally larger than MALDI (Dill et al., 2009).
Despite the ease of ionization, DESI applications focusing on lipids have been limited (Eberlin et al., 2011). The in situ distributions of phospholipids, sphingomyelin and sulfatides have been shown to distinguish disease states in rat, human and canine species (Wiseman et al., 2008; Dill et al., 2009). In plants, the majority of MSI studies employing DESI have focused on analysis of secondary metabolites. Application of DESI to plant tissue containing thick cuticles is difficult due to problems of penetration below the surface, resulting in insufficient signal intensity and stability (Thunig et al., 2011). The same study confronted this limitation by making tissue prints of plant material from leaves and petals of Hypericum perforatum (St John’s wort) and leaves of Datura stramonium (thorn apple), achieving increased signal intensity and sampling reproducibility for identification of a number of secondary metabolites (Thunig et al., 2011). Similar tissue prints on ordinary printer paper were used to image the distribution of the alkaloid malabaricone C in cross-sections of Myristica malabarica Lam. seeds (Ifa et al., 2011). Principal-component analysis of TAGs analyzed in common plant oils (e.g. olive (Olea europaea), safflower (Carthamus tinctorius L.), hazelnut (Corylus L.)) on glass slides by DESI demonstrated a high resolution of MS analysis with limited sample preparation (Gerbig and Takáts, 2010). Recently, glycosides were detected from Stevia rebaudiana leaves with additional limited spectral information possibly corresponding to fatty acids and other lipids (Jackson et al., 2009). A number of additional ambient sampling/ionization MS methods and applications that are currently being developed will probably be available for analysis of plant lipids in the near future (Harris et al., 2011; Liu et al., 2011).