AbstractLattice lightsheet microscopy (LLSM) featuring three-dimensional recording is improved to manipulate cellular behavior with subcellular resolution through optogenetic activation (optoLLSM). A position-controllable Bessel beam as a stimulation source is integrated into the LLSM to achieve spatiotemporal photoactivation by changing the spatial light modulator (SLM) patterns. Unlike the point-scanning in a confocal microscope, the lattice beams are capable of wide-field optical sectioning for optogenetic activation along the Bessel beam path.We show that the energy power required for optogenetic activations is lower than 1 nW (or 24 mWcm^-2) for time-lapses of CRY2olig clustering proteins, and membrane ruffling can be induced at different locations within a cell with subcellular resolution through light-triggered recruitment of phosphoinositide 3-kinase. Moreover, with the epidermal growth factor receptor (EGFR) fused with CRY2olig, we are able to demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable cellular damages.