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BACKGROUND: Two-photon microscopy is widely used to study brain function, but conventional microscopes are too slow to capture the timing of neuronal signalling and imaging is restricted to one plane. Recent development of acousto-optic-deflector-based random access functional imaging has improved the temporal resolution, but the utility of these technologies for mapping 3D synaptic activity patterns and their performance at the excitation wavelengths required to image genetically encoded indicators have not been investigated. NEW METHOD: Here, we have used a compact acousto-optic lens (AOL) two-photon microscope to make high speed [Ca(2+)] measurements from spines and dendrites distributed in 3D with different excitation wavelengths (800-920 nm). RESULTS: We show simultaneous monitoring of activity from many synaptic inputs distributed over the 3D arborisation of a neuronal dendrite using both synthetic as well as genetically encoded indicators. We confirm the utility of AOL-based imaging for fast in vivo recordings by measuring, simultaneously, visually evoked responses in 100 neurons distributed over a 150 μm focal depth range. Moreover, we explore ways to improve the measurement of timing of neuronal activation by choosing specific regions within the cell soma. COMPARISON WITH EXISTING METHODS: These results establish that AOL-based 3D random access two-photon microscopy has a wider range of neuroscience applications than previously shown. CONCLUSIONS: Our findings show that the compact AOL microscope design has the speed, spatial resolution, sensitivity and wavelength flexibility to measure 3D patterns of synaptic and neuronal activity on individual trials.

Original publication




Journal article


J Neurosci Methods

Publication Date





69 - 81


3D functional imaging, Acousto-optic deflectors, Acousto-optic lens, Calcium imaging, Population imaging, Synaptic activity mapping, Two-photon imaging, Action Potentials, Animals, Calcium, Cerebral Cortex, Dendrites, Dendritic Spines, Electroporation, Imaging, Three-Dimensional, In Vitro Techniques, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Multiphoton, Neurons, Patch-Clamp Techniques, Pyramidal Cells, Synapses, Synaptic Transmission, Time, Visual Perception, Voltage-Sensitive Dye Imaging