Three-Dimensional Localization of an Individual Fluorescent Molecule with Angstrom Precision

Furubayashi, Taku; Motohashi, Kazuya; Wakao, Keisuke; Matsuda, Tsuyoshi; Kii, Isao; Hosoya, Takamitsu; Hayashi, Nobuhiro; Sadaie, Mahito; Ishikawa, Fuyuki; Matsushita, Michio M.; Fujiyoshi, Satoru

doi:10.1021/jacs.7b03899

Cited by 15 publications

(18 citation statements)

References 34 publications

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“…However, conventional SR microscopy performed at room temperature is still not considered as a contestant in the arena of structural biology, where Angstrom-level information about the molecular architecture of proteins and protein complexes is sought after. To push the limit of fluorescence microscopy, one can perform measurements under cryogenic conditions (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In addition to slowing down photochemistry, which allows each fluorophore to emit several orders of magnitude more photons than at room temperature (23)(24)(25), a key advantage of cryogenic temperatures is in offering superior sample preservation and high stability for Angstrom-scale structural studies.…”

Section: Introductionmentioning

confidence: 99%

Deciphering a hexameric protein complex with Angstrom optical resolution

Mazal

Wieser

Sandoghdar

2021

Preprint

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Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases, where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and the hexamer geometry of Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic, environmental and dynamic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.Significance statementFluorescence super-resolution microscopy has witnessed many clever innovations in the last two decades. Here, we advance the frontiers of this field of research by combining partial labeling and 2D image classification schemes with polarization-encoded single-molecule localization at liquid helium temperature to reach Angstrom resolution in three dimensions. We demonstrate the performance of the method by applying it to trimer and hexamer protein complexes. Our approach holds great promise for examining membrane protein structural assemblies and conformations in challenging native environments. The methodology closes the gap between electron and optical microscopy and offers an ideal ground for correlating the two modalities at the single-particle level. Indeed, correlative light and electron microscopy is an emerging technique that will provide new insight into cell biology.

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Section: Introductionmentioning

confidence: 99%

Deciphering a hexameric protein complex with Angstrom optical resolution

Mazal

Wieser

Sandoghdar

2021

Preprint

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“…A similar approach was taken by Faoro et al 12 , but this set-up was not applied to super-resolution microscopy. Highly specialized cryo-STORM systems functioning at liquid helium temperature have yielded ~Angstrom localization precision in isolated molecules 13,14 . However, these complex, custom-built cryo-stages have not yet been employed in cell imaging.…”

Section: Introductionmentioning

confidence: 99%

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution

et al. 2019

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Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens ( super SIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, s uper SIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples.

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“…Liu et al [11] attained ~74 nm resolution using DRONPA, a protein 2.5 times brighter than GFP. Recent work [12,13] reported highly specialized cryo-STORM systems functioning at liquid Helium temperature, which yielded an astonishing ~Angstrom localization precision on isolated molecules. Other set-ups relied on custombuilt stages to incorporate cryofluids; Nahmani et al [14] achieved ~35 nm resolution using a waterimmersion objective (NA = ~1.2).…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Bateman

Zanetti-Domingues

et al. 2018

Preprint

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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 cryofixation 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 superresolution 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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Three-Dimensional Localization of an Individual Fluorescent Molecule with Angstrom Precision

Cited by 15 publications

References 34 publications

Deciphering a hexameric protein complex with Angstrom optical resolution

Deciphering a hexameric protein complex with Angstrom optical resolution

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution

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

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