Abstract
Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.
Highlights
To obtain a complete picture of subcellular structures, cells must be imaged in all three dimensions (3D)
A powerful approach to improve imaging in thick cells, the precision of single-molecule localizations, is to reduce the background coming from out-of-focus fluorophores by using light sheet illumination[25,26], where the sample is excited by a thin sheet of light orthogonal to the detection axis
With TILT3D, there is no need to spend considerable effort and expense producing an extremely thin light sheet, because the 3D positions of the single molecules are not determined from the position of the light sheet but instead from the shape of the DH-point spread functions (PSFs)
Summary
To obtain a complete picture of subcellular structures, cells must be imaged in all three dimensions (3D). Early light sheet methods, e.g., selective plane illumination microscopy (SPIM)[25], were designed for low-magnification imaging of large samples, such as embryos These methods are incompatible with imaging close to a coverslip[27] using a high numerical aperture (NA) imaging objective, which is a requirement for high contrast single-molecule imaging of subcellular structures. TILT3D: (a) yields high localization precision of single molecules in 3D over the entire axial range of a mammalian cell via a stack of light sheet slices combined with imaging with engineered PSFs in each slice, (b) has the usual light sheet advantages of reduced photobleaching and photodamage of the sample, and (c) most importantly, is easy and cost-efficient to implement and operate. We validate TILT3D for 3D SR imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the long-range (2 μm) DH-PSF for SM imaging in each slice and the very long-range (6 and 10 μm) tetrapod PSFs for detection of fiducial beads
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