Ultrafast axial scanning for two-photon microscopy via a digital micromirror device and binary holography

In this Letter, we present an ultrafast nonmechanical axial scanning method for two-photon excitation (TPE) microscopy based on binary holography using a digital micromirror device (DMD), achieving a scanning rate of 4.2 kHz, scanning range of similar to 180 mu m, and scanning resolution (minimum step size) of similar to 270 nm. Axial scanning is achieved by projecting the femtosecond laser to a DMD programmed with binary holograms of spherical wavefronts of increasing/decreasing radii. To guide the scanner design, we have derived the parametric relationships between the DMD parameters (i.e., aperture and pixel size), and the axial scanning characteristics, including (1) maximum optical power, (2) minimum step size, and (3) scan range. To verify the results, the DMD scanner is integrated with a custom-built TPE microscope that operates at 60 frames per second. In the experiment, we scanned a pollen sample via both the DMD scanner and a precision z-stage. The results show the DMD scanner generates images of equal quality throughout the scanning range. The overall efficiency of the TPE system was measured to be similar to 3%. With the high scanning rate, the DMD scanner may find important applications in random-access imaging or high-speed volumetric imaging that enables visualization of highly dynamic biological processes in 3D with submillisecond temporal resolution. (C) 2016 Optical Society of America