Fast Multiphoton Microscope Based on Polymer Ulenses Array using a 3D Printed Mold

Multiphoton microscopy is a well-established technique for in vivo tissue imaging at high resolution and long penetration depth. As it uses a single point detector and a single scanning beam, image generation is slow. Several competitive solutions have been proposed during the last years, e.g. resonant scanning, that can reach 30 fps at 2048×2048 scanning resolution but relying on a fixed speed but it does not grant enough time for efficient collection of photons, or multiplexing beams [1,2], which show poor efficiency. Here, we propose a multiplexed beam solution that uses a rotating ulenses array for excitation and a camera for detection. The array of ulenses was built in polydimethilsiloxane (PDMS; > 90% transmission in the near IR wavelengths typically used in multiphoton microscopy), using a 3D-printed mold designed following a Nipkow disc pattern slightly modified to increase the lenses efficiency. This very simple protocol gave us flexibility to improve the array's design based on its performance. The main drawback was the quality of the mold surface obtained with the 3D printer. We surpassed this problem by implementing and intermediate step whereby the mold was spin coated with a very thin layer of PDMS, silanized and then deposited the final PDMS that made the ulens array.