Digital micromirror device-based two-photon microscopy for three-dimensional and random-access imaging

Fast 3D microscopic imaging methods have been playing a crucial role in many biological studies. In this Letter, we present a revolutionary way to design and build a two-photon excitation (TPE) microscope for 3D and random-access imaging based on a digital micromirror device (DMD), achieving a scanning speed of 22.7 kHz. When pairing with a 40× objective lens, the maximum scanning range in the x, y, z axes are 103, 206, 524 μm, respectively. The axial and lateral scanning resolution (i.e., minimum step size) are 270 nm and 130 nm, respectively. In the system, the focal point of the femtosecond laser can be arbitrarily positioned to any random point in space by switching the binary holograms stored in theDMD. Parametric models are derived to deterministically link the DMD parameters (i.e., pixel size and aperture) with the scanner characteristics, i.e., scan range and minimum step size, in each axis. In the experiments, we demonstrate conventional raster scanning, scanning along arbitrarily programmed surfaces, and randomaccess scanning on a pollen grain sample via the DMD-based TPE system. We also perform experiments to demonstrate the unique capability of selective optical stimulation, where selected locations within the specimen are photobleached by extending the laser dwell time. With its versatility and high scanning rate, the TPE microscope may find important applications in brain research, realizing in vivo random-access imaging and optical stimulation with tens of microseconds temporal resolution.