Using rotational integration of oblique interferometric scattering to track axial spatiotemporal responses of tubular membrane protrusions

The 3D spatiotemporal dynamics of tubular membrane protrusions are crucial
for understanding phagocytosis, cellular communication and mechan-
obiology. Confocal microscopy, despite its prevalent use in membrane pro-
trusion 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 inter-
ference 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 sig-
nals 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 volu-
metric imaging.