Combined Scattering, Interferometric, and Fluorescence Oblique Illumination for Live Cell Nanoscale Imaging

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label-free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structures. A combined imaging system would permit obtaining quantitative 3D spatial temporal details of individual cellular components. In this paper, we empirically determined optimal azimuthal scanning angles of rotating beams to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, bright-field (BF) and dark-field (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high-inclined laminated oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering, and fluorescence imaging enable (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of more than ∼1000 platelets suggest that serum albumin (bovine) is necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we identified new asynchronous membrane fluctuations between the leading and rear edges of a migrating platelet. We further demonstrated that interferometric imaging permits the quantification of mitochondrial dynamics on human lung microvascular endothelial cells. Our data suggests that axial displacement of mitochondria is minimal when it is closer to the nucleus or the leading edge of a cell membrane. Taken together, this combined nanoscopy platform helps to quantify multiple spatial temporal events of a migrating cell that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.