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      Fast three-dimensional super-resolution imaging of live cells

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          Abstract

          We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolutions using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast switching probes enabled us to achieve 2D imaging at spatial resolutions of ~25 nm and temporal resolutions as fast as 0.5 sec. We also demonstrated live-cell 3D volumetric super-resolution imaging. A 3D spatial resolution of ~30 nm in the lateral directions and ~50 nm in the axial direction was obtained at time resolutions down to 1 – 2 sec with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we further demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.

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          Most cited references 34

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          Far-field optical nanoscopy.

           Stefan Hell (2007)
          In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
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            Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.

            Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.
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              Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.

               M. Gustafsson (2005)
              Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                1 June 2011
                08 May 2011
                June 2011
                01 December 2011
                : 8
                : 6
                : 499-508
                Affiliations
                [1 ] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138
                [2 ] Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
                [3 ] Department of Physics, Harvard University, Cambridge, MA 02138
                [4 ] Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138
                Author notes
                Correspondence should be addressed to X.Z. ( zhuang@ 123456chemistry.harvard.edu )
                [5]

                These authors contributed equally to this work.

                Article
                nihpa289432
                10.1038/nmeth.1605
                3137767
                21552254

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM068518-06A1 || GM
                Categories
                Article

                Life sciences

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