Wide-field three-dimensional optical imaging using temporal focusing for holographically trapped microparticles

A contemporary challenge across the natural sciences is the simultaneous optical imaging or stimulation of small numbers of cells or colloidal particles organized into arbitrary geometries. We demonstrate the use of temporal focusing with holographic optical tweezers in order to achieve depthresolved two-photon imaging of trapped objects arranged in arbitrary three-dimensional (3D) geometries using a single objective. Trapping allows for the independent position control of multiple objects by holographic beam shaping. Temporal focusing of ultrashort pulses provides the widefield two-photon depth-selective activation of fluorescent samples. We demonstrate the wide-field depth-resolved illumination of both trapped fluorescent beads and trapped HL60 cells in suspension with full 3D positioning control. These approaches are compatible with implementation through scattering media and can be beneficial for emergent studies in colloidal science and particularly optogenetics, offering targeted photoactivation over a wide area with micrometer-precision depth control.