[Fast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level].

INTRODUCTION Two-photon microscopy is the ideal tool to study how signals are processed in the functional brain tissue. However, early raster scanning strategies were inadequate to record fast 3D events like action potentials. AIM The aim of the authors was to record various neuronal activity patterns with high signal-to-noise ratio in an optical manner. METHOD Authors developed new data acquisition methods and microscope hardware. RESULTS Multiple Line Scanning enables the experimenter to select multiple regions of interests, doing this not just increases repetition speed, but also the signal-to-noise ratio of the fluorescence transients. On the same principle, an acousto-optical deflector based 3D scanning microscope has been developed with a sub-millisecond temporal resolution and a millimeter z-scanning range. Its usability is demonstrated by obtaining 3D optical recordings of action potential backpropagation in several hundred micrometers long neuronal processes of single neurons and by 3D random-access scanning of Ca(2+) transients in hundreds of neurons in the mouse visual cortex. CONCLUSIONS Region of interest scanning enables high signal-to-noise ratio and repetition speed, while keeping good depth penetration of the two-photon microscopes.