Imaging cellular network dynamics in three dimensions using fast 3D laser scanning

Abstract

Spatiotemporal activity patterns in three-dimensionally organized cellular networks are fundamental to the function of the nervous system. Despite advances in functional imaging of cell populations, a method to resolve local network activity in three dimensions has been lacking. Here we introduce a three-dimensional (3D) line-scan technology for two-photon microscopy that permits fast fluorescence measurements from several hundred cells distributed in 3D space. We combined sinusoidal vibration of the microscope objective at 10 Hz with 'smart' movements of galvanometric x-y scanners to repeatedly scan the laser focus along a closed 3D trajectory. More than 90% of cell somata were sampled by the scan line within volumes of 250 microm side length. Using bulk-loading of calcium indicator, we applied this method to reveal spatiotemporal activity patterns in neuronal and astrocytic networks in the rat neocortex in vivo. Two-photon population imaging using 3D scanning opens the field for comprehensive studies of local network dynamics in intact tissue.

Enhanced Astrocytic Ca2+Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

“…To test this possibility, we used a cell type selective manipulation that attenuates astrocytic Ca 2ϩ signals. Topical application of AMconjugated BAPTA-based compounds to the cortical surface led to selective labeling of astrocytes (Hirase et al, 2004;Nimmerjahn et al, 2004;Gobel et al, 2007). Astrocytes located hundreds of micrometers beneath the cortical surface label with these compounds (supplemental Fig.…”

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

“…The first is from Gobel et al, who combine a scanning system consisting of galvanometer driven pivoting mirrors for lateral imaging and a piezoelectric actuator for axial imaging with a novel scanning strategy 31 . In essence, this technique represents the furthest extent to which inertia-based scanning techniques can be taken.…”

“…Other techniques which extend the depth of field to provide a maximum projection image of the neuron, such as axicon-based scanning 29,30 , prevent the functional imaging of structures which overlap axially and drastically increase the power requirements for multiphoton imaging. In addition, a recent technique of strategically combining axial movement of the objective lens via a piezoelectric actuator with lateral scanning using galvanometers has been shown to be effective in imaging somatic calcium transient in vivo 31 . However, although this technique employs sinusoidal axial movements, it is still constrained by the inertia of the objective lens, which limits the acquisition rate of the system.…”

Instantaneous three-dimensional sensing using spatial light modulator illumination with extended depth of field imaging

“…galvanometer or AOD scanned laser beam and photomultiplier tube) one can record from multiple sites and planes, but only serially. Sequential scanning of targets is still required, and despite sophisticated scanning systems, these systems cannot monitor multiple points with true simultaneity [8][9][10][11]. One technique that helps alleviate this limitation is to rely upon task-based approaches to imaging and leverage joint optical-digital design strategies that can be used to selectively enhance defocus-related performance through engineering of the optical Point Spread Function (PSF) [2,[12][13][14][15][16].…”