Implementation of 3D Imaging for Two-photon Laser Scanning Microscopy

Information exchange between neural systems occurs at the level of populations of neurons. Thus in order to understand how this information exchange occurs, it is indispensible to understand the role of underlying neuronal systems. Electrophysiological techniques have enhanced our understanding of the nervous system by enabling the study of properties of single ion channels to that of ensembles of neurons. While electrophysiological measurements offer excellent temporal resolution, they lack spatial resolution as this method provides a readout of the electrical signals from single or ensembles of neurons in the vicinity of the electrodes (Scanziani et al, 2009). Imaging techniques have gained a lot of prominence because they are non-invasive and provides excellent spatial resolution (Scanziani et al, 2009). The advent of fluorescent genetically encoded optical probes and other fluorescent synthetic indicators has enabled the study of network functions of neurons (Handel et al, 2008). There are various imaging techniques but the one most suited to study network activity is Multiphoton emission (MPE) microscopy because of its ability to image at greater depths in the tissue. In particular, the most popular and extensively used method in this class is the 2-Photon Microscopy. Imaging method suntil recently have employed 2D scanning at planes normal to the light axis. It is known that processing of information occurs at local ensembles of neurons, hence obtaining population activity in a volume of interest is of greater relevance. This has been possible with the technological advancements over the past couple of years (Gobel et al,2007). The aim of this thesis is to implement a fast 3D scanning algorithm using 2-photon microscopy to measure the activity patterns of neuronal ensembles. Further, this technique could be used in order to relate the activity of neurons with the behavioral output