Study of two-photon Line Excitation Array Detection microscopy

The functional meaning associated with neuronal activity in the mammalian brain is an active area of research limited by the available microscope instrumentation. Exploring this domain of neuroscience necessitates high-speed 3D imaging operating over 1 kHz volumetric scan rates with sub-cellular resolution, as action potentials propagate on sub-millisecond time scales. Monitoring these signals requires in vivo experimentation, so additional care must be taken to avoid invasive methods that may damage sample tissue to live animal subjects. Multi-photon imaging provides an opportunity for non-invasive microscopy with optical sectioning while simultaneously deeply penetrating brain tissue. However, current multi-photon microscopy methods are limited to 10-100 Hz volumetric imaging rates. This thesis explores and expands upon a potential high-speed 2-photon imaging technology, 2-photon Line Excitation Array Detection (2p-LEAD) microscopy. 2p-LEAD combines line scanning with detection via a multi-channel photomultiplier tube (PMT) array, with the potential to operate at 125 kHz frame rates. In the experimental prototype outlined in this thesis, a 1035 nm excitation line of 2.4 µm x 259 µm (1/e2 beam intensity diameter) is scanned at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a fast-scanning galvanometric mirror, we scan the excitation line at 3,000 FPS, generating a 170 µm x 75 µm fluorescence FOV imaged to a 16 x 320 pixel frame. Temporal focusing was implemented to improve optical sectioning and signal-to-noise ratio (SNR), by reducing the out of focus fluorescent signal. This reduction was achieved by dispersing the pulse-width from 300 fs at the focus to multiple picoseconds. 0.5-2 µm fluorescent polystyrene beads were imaged to characterize the system resolution of 1-5.3 µm laterally. Thus this research lays the groundwork for 2p-LEAD imaging at 125 kHz, with an acousto-optic deflector replacing the galvo-mirror as the primary scanning element, for high-speed neuronal imaging.