In vivo live cell imaging for the quantitative monitoring of lipids by using Raman microspectroscopy.

A straightforward in vivo monitoring technique for biomolecules would be an advantageous approach for understanding their spatiotemporal dynamics in living cells. However, the lack of adequate probes has hampered the quantitative determination of the chemical composition and metabolomics of cellular lipids at single-cell resolution. Here, we describe a method for the rapid, direct, and quantitative determination of lipid molecules from living cells using single-cell Raman imaging. In vivo localization of lipids in the form of triacylglycerol (TAG) within oleaginous microalga and their molecular compositions are monitored with high spatial resolution in a nondestructive and label-free manner. This method can provide quantitative and real-time information on compositions, chain lengths, and degree of unsaturation of fatty acids in living cells for improving the cultivating parameters or for determining the harvest timing during large-scale cultivations for microalgal lipid accumulation toward biodiesel production. Therefore, this technique is a potential tool for in vivo lipidomics for understanding the dynamics of lipid metabolisms in various organisms.

Monitoring of cellular behaviors by microcavity array-based single-cell patterning.

In this study, we describe a less invasive and rapid single-cell patterning technique for monitoring of cellular behaviors. To form a high-density grid pattern of living cells, single cells were firstly captured on a geometry-controlled array pattern of 100,000 microcavities by applying negative pressure. The captured cells on the microcavities were immersed in an agarose solution and embedded in agarose gels. The high efficiency transfer of individual yeast cells (Saccharomyces cerevisiae) and diatom cells (Fistulifera sp.) onto agarose gels was successfully achieved in 20 min. The patterning process had no effect on the cell proliferation or division. These results indicate that this technique shows a dramatic increase in patterning efficiency compared to previous patterning technologies. Furthermore, it allows the long-term monitoring of diatom cell divisions for 24 h. Continuous long-term observation of single cells provides technological advantages for the successful acquisition of information to better understand cellular activities.