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Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

By Lin Wang, Benji Bateman, Laura C. Zanetti-Domingues, Amy N. Moores, Sam Astbury, Christopher Spindloe, Michele C. Darrow, Maria Romano, Sarah R. Needham, Konstantinos Beis, Daniel J. Rolfe, David T. Clarke, Marisa L. Martin-Fernandez

Posted 20 Jul 2018
bioRxiv DOI: 10.1101/373647

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve <8 nm localisation precision and 12 nm resolution under cryogenic conditions in a low-cost, low-tech system. This is to our knowledge the best resolution yet attained in biological samples. Furthermore, we demonstrate multicolour imaging and show that the inexpensive setup outperforms 10-fold more costly super-resolution microscopes. By also removing the barrier to total internal reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

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