Abstract

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 light scattering brain tissue requires an imaging/recording mechanism with high spatio-temporal 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 advanced 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 acousto-optic deflectors and does not require axial movements of the objective lens. We have also shown how, when used with the ultra-fast laser pulses needed in multi-photon microscopy, this scheme inherently compensates for the spatial dispersion which would otherwise significantly reduce the resolution of acousto-optic based multi-photon microscopy. We have now coupled 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 (µs) range. The resulting random-access scanning mechanism allows for functional imaging of complex 3D structures such as neuronal dendrites at several thousand volumes per second.

© 2007 SPIE