Construction, Alignment, and Implementation of an Acousto-Optical Deflector-Based System for Patterned Uncaging with Ultraviolet Light

Abstract

The method of patterned photoactivation is a natural fit for the study of neuronal dendritic integration. Photoactivatable molecules that influence a wide range of extracellular and intracellular neurophysiological functions are available. The choice of photosensitive molecules depends on the research question and will influence the design of the experimental apparatus. An acousto-optical deflector (AOD)-based system can be used for rapid ultraviolet (UV) photolysis in arbitrary spatial and temporal patterns. Photolysis-activated “caged” diffusible molecules or newer light-sensitive membrane proteins can be used in this system. This protocol describes the addition of a UV beam for uncaging to a homebuilt two-photon microscope. The goal is to get UV light from the light source (laser) to the approximate center of the objective's back aperture, passing through a pair of perpendicularly oriented AODs along the way. The protocol also describes the fine alignment of the UV beam and the implementation of AOD-based beam steering. Performing the final alignment with the beam passing through the AODs will ensure that the system is optimized for the idiosyncrasies of the crystals.