Initial studies of two-photon line excitation array detection fluorescence microscopy
Creators: name: Wu, Frances Camille Masim
Publisher: name: The University of Texas at Austin
Year: 2020
Subjects: Two-photon microscopy
Light sheet fluorescence microscopy
Neuroscience

description: Rapid, large-scale two-photon fluorescence microscope is essential in volumetric brain imaging. To study the behavior of highly interconnected neuronal circuits and monitor neuronal events i.e. action potentials at millisecond timescales, a two-photon fluorescence microscope with large field-of-view and fast imaging frame rate must be developed. Current advances in two-photon fluorescence imaging techniques have been circumscribed to either a large-scale imaging modality with slow imaging frame rate or a small-scale imaging technique with fast imaging speed. As a result, neuronal investigations are limited to studies in only a fraction of the brain with low spatial and temporal resolutions. To overcome this challenge, this thesis characterizes a custom-built rapid, large-scale two-photon fluorescence microscope, named two-photon line excitation array detection (2P-LEAD) fluorescence microscope. The 2P-LEAD fluorescence microscope combines rapid, line-scanning and fast imaging with a 16-channel photomultiplier tube array to be able to achieve high imaging frame rates. The 2P-LEAD fluorescence microscope studied in this thesis uses a galvanometric scanning mirror which is capable of imaging ~2,600 frames per second. An excitation line with a dimension of ~1 x 24 µm² (x x y 1/e² of intensity beam diameter) is scanned over a ~17 µm field-of-view using a ~1.3 kHz galvanometric mirror. The fluorescence is imaged onto a 16-channel photomultiplier tube in which each channel detects 1/16th of the fluorescence line. The detected fluorescence line is scanned across a ~16-mm active sensor channel length of the photomultiplier tube. A detailed systematic design of the optical parameters and the optimum selection of the optics used in the construction of the 2P LEAD fluorescence microscope are presented. Different microscope characterization techniques such as excitation beam imaging, fluorescence beam imaging, correction for fluorescence signal jittering, and signal-to-noise ratio measurements are also presented. The length of the excitation line at the sample focal plane (~7.7 µm, y, FWHM of intensity square beam diameter) coincides with the de-magnified fluorescence beam at the surface of the 16-channel photomultiplier tube. Fluorescence signal jittering which occurs at ~36.31 µs due to the mismatch between the excitation laser repetition rate and the acquisition frequency of the data acquisition card is corrected using frequency manipulation via the external clock. Furthermore, a signal-to-noise ratio of ~6 is achieved with the developed two-photon LEAD fluorescence microscope. Finally, a design of the future rapid, large-scale 2P-LEAD fluorescence microscope is presented. In the design, an excitation line with a dimension of ~1.5 x 240 µm² (x x y 1/e² of intensity beam diameter) is scanned over a ~100 µm field-of-view using a ~1.3 kHz galvanometric mirror. The fluorescence is detected by a 32-channel photomultiplier tube. To perform fast volumetric imaging of mouse brain in-vivo, a fast-lateral scanning unit i.e. resonant mirror (~24 kHz) or acousto-optic deflector (~MHz) and a fast aberration-free axial remote focusing unit i.e. voice-coil actuator can be incorporated in the custom-built 2P-LEAD fluorescence microscope. The proposed 2P-LEAD can be a promising imaging tool to perform rapid, large-scale mouse brain imaging in-vivo to investigate large volumes of neuronal networks and study neuronal events in millisecond or sub-millisecond timescales.