Browsing by Author "Gagliano, Gabi"

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    Single-Objective Tilted Light Sheet Illumination with Exchange-PAINT and Deep Learning for Fast, Accurate, and Precise 3D Single-Molecule Super-Resolution Imaging in Mammalian Cells
    (2023-05-03) Gagliano, Gabi; Gustavsson, Anna-Karin
    Single-molecule super-resolution fluorescence microscopy is a powerful method for imaging detailed biological structures at the nanoscale. However, imaging capabilities in thick samples such as mammalian cells remain limited due to increased fluorescence background which degrades the achievable localization precision of fluorescent emitters. Light sheet fluorescence microscopy is a simple solution in which a thin plane of light is used to optically section the sample, resulting in an increased signal-to-background ratio and thus improving the achievable localization precision of single molecules. However, light sheet illumination is sensitive to shadowing artifacts from imperfections in the optical path and scattering throughout the sample which may impact the homogeneity of the illumination. Additionally, most light sheet systems employ two objectives, which may suffer from steric hindrance and drift between the sample and illumination objective. In this work, I present a single-objective light sheet microscopy setup which has been combined with the additional innovations of (i) dithering of the light sheet for artifact reduction, (ii) a 3D-printed microfluidic chip for control of the extracellular environment and reflection of the light sheet into the sample, (iii) sequential DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) known as Exchange-PAINT, (iv) engineered point spread functions (PSFs) for 3D imaging, and (v) deep learning for high-density localization of single molecules. Altogether, this approach improves the localization precision, imaging speeds, and multi-target accuracy and enable fast, accurate, and precise multi-target 3D single-molecule super-resolution cellular imaging. First, I introduce single-molecule localization microscopy, light sheet illumination, and the experimental methods of Exchange-PAINT and point spread function engineering which are used in this work. I then describe the design of the light sheet which has been devised with width, thickness, and confocal parameter specifically suited for mammalian cell imaging, and outline the construction and calibration of the optical setup. Finally, I validate the optical system in terms of background reduction, localization precision improvement, and imaging speed by performing multi-target single-molecule super-resolution imaging of nuclear lamina proteins lamin B1, lamin A/C, and emerin, and the microtubule protein alpha-tubulin. The single-objective dithered light sheet achieves a localization precision below ten nanometers in xy and below 12 nanometers in z, enabling a range of applications from nuclear protein investigation to whole cell imaging. I plan to use this system to investigate the relationship between nuclear lamina protein organization and chromatin dynamics in Hutchinson-Gilford Progeria Syndrome (HGPS), a disorder caused by a mutation in the LMNA gene.