Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity

Abstract

The dynamic ability of neuronal dendrites to shape and integrate synaptic responses is the hallmark of information processing in the brain. Effectively studying this phenomenon requires concurrent measurements at multiple sites on live neurons. Significant progress has been made by optical imaging systems which combine confocal and multiphoton microscopy with inertia-free laser scanning. However, all systems developed to date restrict fast imaging to two dimensions. This severely limits the extent to which neurons can be studied, since they represent complex threedimensional (3D) structures. Here we present a novel imaging system that utilizes a unique arrangement of acousto-optic deflectors to steer a focused ultra-fast laser beam to arbitrary locations in 3D space without moving the objective lens. As we demonstrate, this highly versatile randomaccess multiphoton microscope supports functional imaging of complex 3D cellular structures such as neuronal dendrites or neural populations at acquisition rates on the order of tens of kilohertz.

Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach

“…For example, acousto-optic methods of fast 3D laser-scanning allow random-access scanning patterns, in which only a subset of voxels of interest is sampled, and can sharply increase the sampling duration per voxel as compared to a raster-scan of the total volume of interest (Duemani Reddy et al, 2008; Grewe et al, 2010; Katona et al, 2012). Although acousto-optic scanners have some intricate optical drawbacks, one simple drawback when studying behaving mammals is that random-access scanning approaches neither are robust to brain motion nor produce data sets conducive to image alignment.…”

Automatic Identification of Fluorescently Labeled Brain Cells for Rapid Functional Imaging

“…The relatively high efficiency of this process may, in some instances, obviate the need to replace galvometric scanners with acousto-optical deflectors (AODs). Arbitrary path scanning can also be combined with AODs for three-dimensional scanning applications (Duemani-Reddy et al 2008;Vucinić and Sejnowski 2007).…”

A Guide to Emerging Technologies for Large-Scale and Whole-Brain Optical Imaging of Neuronal Activity

“…One class of Ca 2+ imaging that exploits spatial sparseness and uses prior knowledge on neuronal positions is RAM (Fernández-Alfonso et al 2014, Katona et al 2012, Reddy et al 2008). In this modality, fast scanners, such as AODs, are used to access points in 3D that are known to contain neurons or neuronal processes along arbitrary trajectories in the volume (Fig.…”