Microalgae are promising candidates for biofuel production due to their high lipid content. To facilitate utilization of the microalgae for biofuel, rapid quantification of the lipid contents in microalgae is necessary. However, conventional methods based on the chemical extraction of lipids require a time-consuming destructive extraction process. Here, we demonstrate label-free, non-invasive, rapid quantification of the lipid contents in individual micro-algal cells measuring the three-dimensional refractive index tomograms. We measure three-dimensional refractive index distributions within Nannochloropsis oculata cells and find that lipid droplets are identifiable in tomograms by their high refractive index. In addition, we alter N. oculata under nitrogen deficiency by measuring the volume, lipid weight, and dry cell weight of individual cells. Characterization of individual cells allows correlative analysis between the lipid content and size of individual cells.

Biofuels have attracted increasing attention as sustainable energy resources that could potentially replace fossil fuels. Among the candidates suggested for biofuel production are a variety of plants 1, 2 , fungi 3 , bacteria 4, 5 , and microalgae 6 . The latter has long been considered a promising sustainable resource due to their rapid growth and high lipid content 7,8 . Extensive research has been conducted to find suitable species and to optimize cultivation conditions to make large-scale lipid production by microalgae economically feasible 9, 10 . Even after this attention, there is still a high demand for development of new strains and optimized cultivation processes to facilitate industrialization of microalgae-based biofuels.To enhance the research and development of microalgae strains for biofuel applications, it is essential to develop rapid, quantitative methods for lipid detection. Conventionally, gravimetric methods and chromatography have been widely used to quantify lipid content in microalgae. Because these methods are based on chemical extraction of the lipids using organic solvents; however, the methods require laborious, time-consuming extraction processes that take from several hours to days. Furthermore, the destructive nature of the extraction leads to irreversible loss of a large volume of each sample, which restricts repeated measurements. As an alternative, lipid quantification based on microscopic imaging techniques has recently been proposed in conjunction with advances in the relevant imaging techniques and instruments. Compared with quantification based on chemical extraction, quantification via imaging has the advantages of speed and low sample consumption. For example, fluorescence microscopy employing lipophilic fluorescent dyes is a representative imaging technique for visualizing and quantifying the lipid content in individual cells 11,12 . Unfortunately, the fluorimetric methods are limited in principle to qualitative results because the permeability of cell membranes to dyes varies among microalgae species and the dye solutions used

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