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

Abstract

ABSTRACTTo 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 structure. In doing so, a combined imaging system would permit quantitative 3D spatial temporal detail of individual cellular components. In this paper, we empirically determined a series of optimal azimuthal scanning angles and rotating beam to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, brightfield (BF) and darkfield (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 enables (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 adherent endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of over ∼1000 platelets, suggested that serum albumin (bovine) was necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we determine new asynchronous membrane fluctuations between the leading and rear edge of a migrating platelet. We further demonstrated that interferometric imaging permitted the quantification of mitochondria dynamics on lung microvascular cells (HMVEC). Our data suggests that axial displacement of mitochondria is minimized when it is closer to the nucleus or the leading edge of a cell membrane that exhibits retrograde motion. Taken together, this combined imaging platform has proven to quantify multiple spatial temporal events of a migrating cell, that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.