Conventional methods of solvent extraction and gravimetric determination for lipid quantification (Bligh and Dyer, 1959) are laborious and time consuming. Moreover, approximately 10 to 15 mg wet weight of cells (Akoto et al., 2005) must be cultured for any appreciable extraction and derivatization. However, in-situ lipid content mea­surements would significantly reduce the quantity of sample as well the preparation time required. Accordingly, there is greater interest in a rapid in-situ measurement of the lipid content of algal cells (Cooksey et al., 1987). Nile Red (9-diethylamino — 5H-benzo[a]phenoxazine-5-one), a lipid-soluble fluorescent dye, has been com­monly used to evaluate the lipid content of animal cells and microorganisms such as yeasts and fungi (Genicot et al., 2005) and specifically extended to microalgae (Cooksey et al., 1987; Elsey et al., 2007). Nile Red is relatively photostable and pro­duces intense fluorescence in organic solvents and hydrophobic environments, which makes them a better candidate for in-situ screening for lipids. Furthermore, neutral and polar lipids can be clearly differentiated due to polarity changes in the medium as evinced by a blue shift in the emission maximum of Nile Red (Greenspan and Fowler, 1985; Laughton, 1986; Cooksey et al., 1987; Lee et al., 1998). The solvent system used for Nile Red would determine the emission spectra of the dye (Elsey et al., 2007). However, the thick cell walls of microalgae inhibit the permeation of Nile Red, and this is variable among algal species, requiring the use of high levels of solvents such as DMSO (20% to 30% v/v) and elevated temperatures (40°C) (Chen et al., 2009). Then again, Chen et al. (2011) developed a two-step microwave-assisted staining method for in vivo quantification of neutral lipids in green algae with thick, rigid cell walls that prevents penetration of the Nile Red dye into the cell. This may also be appropriate for other classes of algae that do not stain properly with Nile Red. Hence, a Nile Red assay can be used as a tool for screening oleaginous algal strains as well as quantitatively determining the neutral lipids in algal cells (Figure 3.2; see color insert).

Recently, another class of lipophilic fluorescent dye BODIPY® 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) has been used to potentially stain microalgal lipids. BODIPY staining lets the lipid droplets stain green and the chloroplasts stain red in live algal cells (Cooper et al., 2010). BODIPY 505/515 is advantageous over Nile Red in emitting a narrower spectrum (Cooper et al., 2010;

Govender et al., 2012). This facilitates the fluorescence distinction of lipid bodies, resulting in better resolution and thus is important for seamless confocal imaging (Cooper et al., 1999). Furthermore, unlike Nile Red, BODIPY 505/515 does not fix to cytoplasmic constituents other than lipid bodies and chloroplasts. This discerning property of BODIPY 505/515 to bind to lipid bodies alone offers rapid screening and isolation of hyper-lipid producing algal strains. Bigelow et al. (2011) developed a rapid, single-step, laboratory-scale in-situ protocol for GC-MS (gas chromatogra­phy with mass spectroscopy) lipid analysis that requires only 250 qg dry mass per sample. When coupled with fluorescent techniques using Nile Red or BODIPY dyes and flow cytometry for cell sorting, the aforesaid GC-MS analysis allows throughput screening of lipid-producing algal strains from varied environments. Upon isolation, purification, and identification of a hyper-lipid producing algal strain, the researcher would be interested in the physiological traits such as the photosynthetic efficiency, carbon fixation rate, growth rate, etc. Alternatively, infrared analysis, which does not depend on stain application but rather detects specific molecular absorption bands to give approximate concentrations, can be used for the detection of many metabolites, including lipids. This method has recently been applied to detecting changes in algal cell composition during nitrogen starvation (Dean et al., 2010).

Spectroscopic methods such as near-infrared (NIR) and Fourier transform infra­red (FTIR) spectroscopies have been established to predict the levels of spiked polar and neutral lipids in algal cells based on multivariate calibration models (Laurens and Wolfrum, 2011). The above infrared spectroscopic techniques are rapid, high — throughput, and non-destructive means of algal screening for lipids. Hence, this cal­ibration model serves as a short-time, high-throughput method of quantifying cell lipids compared to time-consuming traditional wet chemical methods. The NIR and FTIR spectra of biomass of various species accurately predicted the levels of lipids. This fast, high-throughput spectroscopic lipid fingerprinting method is pragmatic in real-time monitoring of lipid accumulation or a multitude of screening efforts that are ongoing in the microalgal research community. Coherent anti-Stokes Raman scatter­ing microscopy is also an associated technique that creates an image of whole cells based on the vibrational spectra of a specific cellular constituent. Huang et al. (2010) demonstrated that Stokes Raman spectroscopy could accomplish detection and identi­fication of cellular storage lipids, specifically triglycerides. Further, similar to infrared spectroscopic techniques, Raman scattering microscopy is also prospective as a rapid, noninvasive compositional analysis method that enables imminent in-line or at-line lipid monitoring. Recently, a single-cell, laser-trapping Raman spectroscopic method that is direct and in vivo has been described as an efficient tool for profiling microbial cellular lipids (Wu et al., 2011). This method is proven in the quantitative estimation of the degree of unsaturation and transition temperatures of algal cellular lipids.