Development of a random access multiphoton microscope for fast three-dimensional functional recording of neuronal activity

Duemani Reddy; Peter Saggau

doi:10.1117/12.701077

14 February 2007 Development of a random access multiphoton microscope for fast three-dimensional functional recording of neuronal activity

Duemani Reddy, Peter Saggau

Author Affiliations +

Proceedings Volume 6443, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIV; 64430U (2007) https://doi.org/10.1117/12.701077
Event: SPIE BiOS, 2007, San Jose, California, United States

Abstract

Over the past two decades, the dendritic processes of neurons have been shown to possess active and dynamic properties that give them the ability to modulate synaptic integration and shape individual synaptic responses. Effectively studying these properties at multiple locations on a live neuron in highly scattering brain tissue requires an imaging/recording mechanism with high spatiotemporal resolution as well as optical sectioning and random access site selection capabilities. Our lab has made significant steps in developing such a system by combining the spatial resolution and optical sectioning ability of imaging techniques such as confocal and multi-photon microscopy with the temporal resolution and random access capability provided by acousto-optic laser scanning. However, all systems that have been developed to date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which many neurons can be studied since they represent complex three-dimensional (3D) structures. We have previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of multiple acousto-optic deflectors and does not require axial movements of the objective lens. Here we couple this scanning scheme to a modified commercial research microscope and use the combined system to effectively image user-defined sites of interest on fluorescent 3D structures with positioning times that are in the low microsecond range. The resulting random-access scanning mechanism allows for functional imaging of complex 3D cellular structures such as neuronal dendrites at frames rates on the order of tens of kilohertz.

Citation Download Citation

Duemani Reddy and Peter Saggau "Development of a random access multiphoton microscope for fast three-dimensional functional recording of neuronal activity", Proc. SPIE 6443, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIV, 64430U (14 February 2007); https://doi.org/10.1117/12.701077

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