The 3D spatiotemporal dynamics of tubular membrane protrusions are crucial for understanding phagocytosis, cellular communication and mechanobiology. Confocal microscopy, despite its prevalent use in membrane protrusion studies, presents limitations due to its inherently low axial resolution and high phototoxicity, which significantly hinder live imaging of tubular protrusion along the axial plane. We discovered that rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon generates speckle-free widefield interferometric scattering signals with a 10-fold signal-to-noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free imaging of diverse nanoparticles and tubular membrane protrusions, thus providing biophysical profiling of tubular membrane protrusions across multiple cell types and in complex co-cultures. RO-iSCAT empowers rapid quantitative dissection of the axial spatiotemporal complexities of membrane protrusions at tens to hundreds of nanometer displacements without requiring 3D volumetric imaging. The authors develop RO-iSCAT, which uses rotational integration of off-axis oblique-illuminated interference scattering signals to remove speckle noise in real time. It enables fast, label-free imaging of axial spatiotemporal dynamics of tubular membrane protrusions.
Liu et al. (Thu,) studied this question.