Selective activation of photoactivatable fluorescent protein based on binary holography.

The ability to deliver laser doses to different target locations with high spatial and temporal resolution has been a long-sought goal in photo-stimulation and optogenetics research via, for example, photoactivatable proteins. These light-sensitive proteins undergo conformational changes upon photoactivation, serving functions such as triggering fluorescence, modulating ion channel activities, or initiating biochemical reactions within cells. Conventionally, photo-stimulation on light-sensitive proteins is performed by serially scanning a laser focus or via 2D projection, which is limited by relatively low spatiotemporal resolution. In this work, we present a programmable two-photon stimulation method based on a digital micromirror device (DMD) and binary holography to perform the activation of photoactivatable green fluorescent protein (PAGFP) in live cells. This method achieved grayscale and 3D selective PAGFP activation with subcellular resolution. In the experiments, we demonstrated the 3D activation capability and investigated the diffusion dynamics of activated PAGFP on the cell membrane. A regional difference in cell membrane diffusivity was observed, indicating the great potential of our approach in interrogating the spatiotemporal dynamics of cellular processes inside living cells.