Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 109; no. 3; pp. 661 - 662
Main Authors Rego, E. Hesper, Shao, Lin, Macklin, John J., Winoto, Lukman, Johansson, Göran A., Kamps-Hughes, Nicholas, Davidson, Michael W., Gustafsson, Mats G. L.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 17.01.2012
National Acad Sciences
SeriesPNAS Plus
Subjects
Actin Cytoskeleton - metabolism
Animals
Biological samples
Biological Sciences
Cells
Cells - metabolism
CHO Cells
Cricetinae
Cricetulus
Cytoskeleton
Fluorescence
HEK293 Cells
Humans
image analysis
Light
light intensity
light microscopes
Luminescent Proteins - metabolism
Mammals
microfilaments
Microscopy
Microscopy - methods
microtubules
Microtubules - metabolism
Nonlinear Dynamics
nuclear membrane
Nuclear Pore - metabolism
Photobleaching
PNAS Plus
PNAS PLUS (AUTHOR SUMMARIES)
Polystyrene
Proteins
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Summary:Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.
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Author contributions: M.G.L.G. led the project and conceived of the idea; E.H.R. and M.G.L.G. designed research; E.H.R. performed research; E.H.R., L.S., N.K.-H., and M.W.D. contributed reagents/analytic tools; L.S. wrote the reconstruction software and edited the paper; J.J.M. performed a crucial photobleaching experiment; L.W., G.A.J., and E.H.R. built optical hardware; E.H.R. and M.G.L.G. analyzed data; and E.H.R. wrote the paper.
3Deceased April 17, 2011.
1Present address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115.
Edited by* Jennifer Lippincott-Schwartz, National Institutes of Health, Bethesda, MD, and approved October 18, 2011 (received for review May 16, 2011)
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.1107547108