Combined Afm And Vertical Light Sheet Microscopy To Correlate Actin Accumulation To Engulfment Forces During Phagocytosis

We have combined a vertical light sheet, single objective system that provides fast side-view volumetric imaging simultaneous with AFM (AFM-LS) for mechanobiology applications. The employment of fast steering mirrors and a high aspect ratio, low phototoxic Line Bessel light sheet uniquely poises our system to capture both sub-second and long-term cellular processes. The implementation of a reflecting prism provides sufficient light collection of the selectively illuminated XZ-plane as well as convenient access for spherical, opsonized force probes. With this instrument we demonstrate side-view and volumetric imaging of individual cells to reveal the dynamic accumulation of filamentous actin around a target of phagocytosis; the immune cell process to uptake harmful particles and debris. Furthermore, we enhanced the clarity of the target-cup interface by attaching an index matched, optically clear polyacrylamide bead to the AFM cantilever. In addition to removing the optical lensing effect of traditional spherical probe tips, these antibody-coated polyacrylamide beads are deformable, allowing for quantitative analysis of protein concentrations during bead uptake. With this technique, we have captured high resolution, volumetric imaging of phagocytosis induced actin remodeling concurrent with the associated engulfment forces on the AFM probe. Our application of AFM-LS to phagocytosis revealed target introduced downward forces in excess of 100pN, sufficient for mechanoreceptor induced signaling and significant changes in target shape. Correlation analysis of the engulfment forces to the actin intensity in distinct macrophage cell regions revealed a positive correlation between actin accumulation and a downward force before cup formation. This strongly suggests that phagocytosis is regulated by mechanotransductive signaling within the cup, at separate basal contacts, and potentially within the nucleus. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.