Pathogen-mediated activation of macrophages upregulates dorsal ruffling at the cell surface, which in turn enhances the cells’ capacity for macropinocytosis and phagocytosis. Dorsal ruffles also contribute to receptor signaling and inflammatory responses by housing Toll-like receptors and their associated signaling machinery. High speed, high resolution 4D imaging of live macrophages with the lattice light sheet microscope (LLSM) generates unique insights into cell behaviours over extended periods of imaging, with the potential for extensive quantification in the acquired movies. LLSM imaging is used here to demonstrate and analyze the dynamic nature of dorsal ruffling and it reveals a new form of ruffles induced by LPS. LPS-induced ruffles have a novel structure and give rise to enlarged macropinosomes. A series of Rab GTPases involved in the formation of ruffles, their transition to macropinosomes and TLR signaling has been identified. LLSM detects ruffling defects associated with CRISPR deletion of single Rabs.  As a new imaging modality, LLSM has thus uncovered a new cell surface structure with key roles in pathogen-activated macrophages.