3D imaging lipidometry in single cell by in-flow holographic tomography

Abstract

The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characterization in terms of number, size, spatial positioning and relative distribution in the cell volume can shed light on the roles played by LDs. Until now, fluorescence microscopy and transmission electron microscopy are assessed as the gold standard methods for identifying LDs due to their high sensitivity and specificity. However, such methods generally only provide 2D assays and partial measurements. Furthermore, both can be destructive and with low productivity, thus limiting analysis of large cell numbers in a sample. Here we demonstrate for the first time the capability of 3D visualization and the full LD characterization in high-throughput with a tomographic phase-contrast flow-cytometer, by using ovarian cancer cells and monocyte cell lines as models. A strategy for retrieving significant parameters on spatial correlations and LD 3D positioning inside each cell volume is reported. The information gathered by this new method could allow more in depth understanding and lead to new discoveries on how LDs are correlated to cellular functions. 3D D. Pirone, P. Memmolo, V. Bianco, L. Iom- marini and P. Ferraro analyzed and discussed the tomographic reconstruc-tions and data. I. Kurelac, S. Lemma and L. Iommarini performed FM ex- periments and data analysis; G. Pasquinelli and S. Valente performed the TEM; all the authors contributed to critical discussion of the results and contributed to write the manuscript. P. Ferraro supervised the research.