| Light sheet microscopy, with the technique developed nearly a century ago and the first application toward fluorescence microscopy of biological specimens occurring in the late twentieth century, has seen a resurgence for applications requiring rapid or 3D imaging of biological samples. Conventional light sheet microscopy uses Gaussian beams that are the standard output of laser systems, and can be turned into a light sheet utilizing a cylindrical lens. However, highly focused Gaussian beams used in microscopy spread out quickly, leaving only a small section that can be imaged, and are also vulnerable to beam propagation distortion and steering in a scattering medium. In order to overcome the depth-of-focus versus resolution tradeoff inherent to Gaussian beams, non-diffracting beams have been proposed as solutions in applications where cellular resolution is required over a larger field-of-view (FOV). These non-diffracting beams, such as Bessel or Airy beams, offer significant improvements in depth-of-focus, but come with disadvantages, such as out-of-focus excitations that degrade contrast and image quality in fluorescent microscopy. However, due the interest in achieving higher FOVs without sacrificing resolution, there is a great deal of ongoing research looking at side lobe suppression techniques with non-diffracting beams.;In this thesis work we look at various methods of suppressing the side lobes of the Bessel beams and assess the contrast differences in comparison to a conventional Gaussian beam. Using a spatial light modulator and dual-axis microscope architecture, we create a test bed for beam shaping and comparing traits between the beam profiles. Differences in image contrast and signal-to-background ratio (SBR) are assessed when looking at fluorescent solutions, beads, and phantoms. In the final section, we look at preliminary experiments on using the sectioned Bessel beam with electronic confocal slit detection (eCSD) image processing and analyze the benefits, along with challenges faced when applying this technology to imaging cleared tissue specimens. Overall, the sectioned Bessel beam coupled with eCSD image processing allows us to achieve similar signal-to-background ratios (SBR) compared to a conventional Gaussian beam illumination scheme, while giving large improvements in depth-of-focus for a given resolution. Although we are unable to prove this in the current iteration of the microscope test bed, these properties are expected to be particularly useful in imaging cleared tissue samples. |
