1. Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.
- Author
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Szalai AM, Siarry B, Lukin J, Williamson DJ, Unsain N, Cáceres A, Pilo-Pais M, Acuna G, Refojo D, Owen DM, Simoncelli S, and Stefani FD
- Subjects
- Animals, COS Cells, Cells, Cultured, Chlorocebus aethiops, DNA metabolism, Fibroblasts cytology, Fibroblasts metabolism, HeLa Cells, Humans, Image Processing, Computer-Assisted methods, Mice, Microtubules metabolism, Photometry methods, Fluorescence, Imaging, Three-Dimensional methods, Microscopy, Fluorescence methods, Nanotechnology methods, Single Molecule Imaging methods
- Abstract
Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.
- Published
- 2021
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