ABSTRACT
One of the most challenging questions in neuroscience is how
neuronal circuits in different brain areas process incoming stimuli
such as tones (auditory cortex), contrasts (visual cortex), or
touch (somatosensory cortex). While working principles of such
neuronal networks can be investigated to some extent by imaging
cultured cell networks and acute brain slice preparations, gaining
a more complete real-world picture requires monitoring of tens or
hundreds of neurons in the intact brains of living animals. Because
most neurons are located relatively deep below the brain surface
(>100 μm), optical measurement techniques used have to provide a
sufficient imaging depth, combined with the ability to resolve the
activity of individual cells within a neuronal network. Two-photon
laser scanning microscopy meets these criteria and has become the
standard technique for functional in vivo measurements of neuronal
populations. In addition, because communication between neuronal
cells usually happens on a time scale of tens of milliseconds,
it is also desirable to record neuronal activity at similar or
even higher sampling rates. This chapter therefore focuses on a
recent technological development that uses acousto-optic deflectors
(AODs) as laser scanners to improve the scanning speed when
imaging large areas of neuronal cell networks. Beginning with the
underlying technical basics of two-photon microscopy, acousto-
optics, and laser beam dispersion issues, important advantages
and disadvantages of the novel high-speed scanning method are
discussed, followed by a description of possible cell-scanning
methods. Finally, an example of an in vivo imaging experiment from
mouse visual cortex is presented to demonstrate the advantages
of the AOD system for high-speed scanning for in vivo two-photon
microscopy.
