Light-sheet microscopy used for tracking particles
Fluorescence microscopy is one of the cornerstones of modern biology but has generally been limited to 2D culture dishes. Light-sheet microscopy, a recent advance which was awarded Nature Method of the Year in 2014, allows fast, non-invasive 3D imaging across an entire organism. This works by decoupling illumination and detection such that the microscope only illuminates a thin section of tissue at a time. By scanning this light-sheet through an organism we can image in 3D, more quickly and with less damage than other techniques such as confocal microscopy. In this work, a custom digitally scanned light-sheet microscope was built, for which the technology was applied and developed to enable two biological studies: the study of material properties of developing embryos and the tracking of virus particles in live cells. In addition to designing and constructing a light-sheet fluorescence microscope, several technological improvements were also investigated to better address these biological questions. The first was a three-dimensional region-of-interest technique which greatly simplifies volumetric imaging calibration whilst also being more robust, with an observed 42% improvement in light collection efficiency compared to current approaches. The projective mathematical theory, used in this technique, was then applied to optical projection tomography to produce a new triangulation-based reconstruction algorithm that is robust to affine sample motion, including mechanical jitter and systematic drift. The second improvement for light-sheet microscopy builds upon confocal slit scanning, a technique used to increase image contrast whilst doubling the acquisition time for a single image. By exploiting the acquisition procedure for confocal slit scanning, full speed imaging with the same increased contrast was realised. Finally an open-hardware solution for multi-scale sample mounting was produced. These improvements to speed, contrast and acquisition speed in the light-sheet microscope allowed us to address the biological questions of interest.
light-sheet, microscopy, pose-estimation, optical projection tomography, particle tracking, spt, fluorescence
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