Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.We acknowledge support from the National Institute of Health 5-P41-EB015871-28, 2R01EY017656-06, R21-NS091982-01, 1-U01-NS090438-01, 1U01CA202177-01, and 1R01HL121386-01A1, Singapore-MIT Alliance for Science and Technology, Connecticut Children’s Medical Center, Hamamatsu Corp., and Samsung Advanced Institute of Technology. C.J.R. further acknowledges support from the Wellcome Trust 093831/Z/10/Z