Microalgae have been identified as a promising renewable feedstock for production of lipids for feeds and fuels. Current methods for identifying algae strains and growth conditions that support high lipid production require a variety of fluorescent chemical indicators, such as Nile Red and more recently, Bodipy. Despite notable successes using these approaches, chemical indicators exhibit several drawbacks, including non-uniform staining, low lipid specificity, cellular toxicity, and variable permeability based on cell-type, limiting their applicability for high-throughput bioprospecting. In this work, we used in vivo hyperspectral confocal fluorescence microscopy of a variety of potential microalgae production strains (Nannochloropsis sp., Dunaliella salina, Neochloris oleoabundans, and Chlamydomonas reinhardtii) to identify a label-free method for localizing lipid bodies and quantifying the lipid yield on a single-cell basis. By analyzing endogenous fluorescence from chlorophyll and resonance Raman emission from lipid-solubilized carotenoids we deconvolved pure component emission spectra and generated diffraction limited projections of the lipid bodies and chloroplast organelles, respectively. Applying this imaging method to nutrient depletion time-courses from lab-scale and outdoor cultivation systems revealed an additional autofluorescence spectral component that became more prominent over time, and varied inversely with the chlorophyll intensity, indicative of physiological compromise of the algal cell. This signal could result in false-positives for conventional measurements of lipid accumulation (via spectral overlap with Nile Red), however, the additional spectral feature was found to be useful for classification of lipid enrichment and culture crash conditions in the outdoor cultivation system. Under nutrient deprivation, increases in the lipid fraction of the cellular volume of ~. 500% were observed, as well as a correlated decrease in the chloroplast fraction of the total cellular volume. The results suggest that a membrane recycling mechanism dominates for nutrient deprivation-based lipid accumulation in the microalgae tested.