An addressable confocal microscope for functional imaging of neuronal activity
Doctor of Philosophy
The study of computation occurring in single neurons and small networks of interconnected neurons is often limited by (1) the number of sites that can be simultaneously probed with electrophysiology tools such as patch pipettes and (2) the recording speed of fluorescence imaging tools such as confocal or multiphoton microscopy. Even in the line scan mode of galvanometer-based scanners, where one scan dimension is sacrificed to gain overall speed, the effective frame rate is limited to less than 1 kHz with no flexibility in site selection. To overcome these limitations and allow the study of many sites throughout the dendritic arbor, we have developed an addressable confocal laser-scanning microscope that permits recording from user-selected sites-of-interest at high frame rates, in addition to conventional full frame imaging. Our system utilizes acousto-optic deflectors (AODs) in the illumination pathway to allow for rapid user-defined positioning of a focused laser spot. However, since AODs rely on diffraction to steer a laser beam, they cannot effectively descan the fluorescence emission spectrum as done in mirror-based systems which utilize reflection; this prevents the use of a stationary pinhole as a spatial filter. Instead, we implement an addressable spatial filter using a digital micromirror device (DMD) in conjunction with the AODs to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current version of the confocal system has a spatial resolution of ∼1 mum. Furthermore, by letting the user tailor which sites are visited, we have shown that recordings can be made at an aggregate frame rate of ∼40 kHz. We have successfully demonstrated that the system is capable of optical sectioning and thus exhibits the main advantage of a confocal microscope for light-scattering biological tissue. This property was used to create three-dimensional reconstructions of fluorescently labeled test specimens. Additionally, we have used the system to record intracellular calcium transients using the fluorescent calcium indicator Oregon Green BAPTA-1. The transients were a result of back-propagating action potentials elicited via 1 nA current injections in cultured hippocampal neurons from wild-type mice.
Neurosciences; Biomedical engineering; Optics