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WIREs Nanomed Nanobiotechnol
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Nanoscopy for nanoscience: how super‐resolution microscopy extends imaging for nanotechnology

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Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence‐based super‐resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single‐molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super‐resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches. WIREs Nanomed Nanobiotechnol 2015, 7:266–281. doi: 10.1002/wnan.1300 This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale
Resolution basis and implications in light microscopy. (a) Airy disk diffraction pattern produced by optical focusing of light to produce an image of a single point‐based object. (b) Contrast produced by different separations of two‐point objects. Three images are shown with linescan intensity plots showing the range of intensities across the middle of each image. Only when the objects are separated beyond the point where the first maxima and minima coincide (Rayleigh criteria, middle diagram, and graph) is contrast and resolvability produced. (c) The Rayleigh formula describing the resolution in conventional fluorescence microscopy. The resolution is determined by the wavelength of light and the numerical aperture (NA) of collection optic. 450‐nm blue light with a 1.4 NA immersion objective gives theoretical resolution of ∼200 nm. (d) Schematic representation of the ability to detect objects smaller than the resolution with fluorescence‐based microscopy. The images of all beads 200 nm and smaller appear the same size. Source: (a and b) adapted from Wikipedia.
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Uses of super‐resolution microscopy with nanotechnology. (a) Schematic view of an approach for imaging nanocatalysts in a flow chamber where the substrate is converted to a fluorescent product. Inset shows scanning electron microscopy (SEM) image of an individual nanocatalyst. (b) The typical image of an individual molecule and the positioning accuracy of the fit. (c) Summed positioning of visualized and positioned individual catalytic events fitted to bins of 20 × 20 nm. (a–c: Reprinted with permission from Ref . Copyright 2012 Macmillan Publishers Ltd). (d) Summed wide‐field fluorescence of Dronpa bound to a nanowire. (e) The summed density of positioning single molecules. (f) High‐resolution imaging of the distribution of protein label over the surface of the nanotriangle. (d–f: Reprinted with permission from Ref . Copyright 2012 John Wiley and Sons)
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Single‐molecule positioning methods. (a) The centroid position of an isolated molecule producing a diffraction‐limited image can be positioned (red circle) much more accurately than the scale of the point‐spread function (green circle). By sequential imaging and positioning isolated molecules, the summed positions of many thousand molecules produce a high‐resolution image. (b) Example of the resolution improvement obtained in an image of a cell expressing a lysosomal marker. The left figure shows the conventional resolution of a TIRF (total internal reflection fluorescence) image, the right panels show zoomed images of the super‐resolution PALM (photoactivated light microscopy) image. (Reprinted with permission from Ref . Copyright 2006 AAAS). (c) 3D super‐resolution image produced by interferometric PALM. Color coding shows the axial positioning of membrane‐targeted fluorescent protein in a mammalian cell; lower figures show an enlarged area and orthogonal view. (Reprinted with permission from Ref . Copyright 2009 National Academy of Sciences, USA)
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The means of generation of improved resolution with stimulated emission depletion (STED). (a) After conventional excitation, certain fluorophores can be emission‐depleted by illumination at the depletion wavelength. Instead of productive fluorescence emission (λem), the energy is dissipated via a different transition releasing a photon outside the detection range of the imaging system (λSTED). (b) By spatial alignment of the excitation and depletion beams, the resulting region of effective excitation and the resulting PSF (point‐spread function) is made smaller and consequently a higher resolution raster‐based image can be produced. (Reprinted with permission from Ref . Copyright 2006 Macmillan Publishers Ltd). (c) When using multiple fluorescence labels, the longer depletion wavelength needs to not overlap with the excitation spectra of any of the fluorophores. One means of achieving this is using fluorophores of distinct excitation and emission and use a long wavelength laser (λSTED) that offers adequate depletion for all dyes. (d) An example of the difference in resolution that can be achieved with STED and standard confocal microscopy. A mixture of 20‐ and 40‐nm fluorescent beads were imaged with confocal, STED, and atomic force microscopy (AFM). Inset figure highlights regions in STED (top), AFM (middle), and confocal (bottom). The higher resolution of STED gives improved ability to resolve closely spaced particles and distinguish particles by size. STED offers similar abilities to a physical process unlimited by diffraction—AFM. (Reprinted with permission from Ref . Copyright 2013 Creative Commons license)
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Structured illumination microscopy (SIM) basis, implementation, and example output. (a) Interference between two high‐frequency patterns produces a lower frequency result. Two high‐frequency grid patterns are inclined to each other by a small angle and the two broader bands running vertically are produced and apparent. If high‐frequency patterned illumination is projected onto a sample, interference with the intrinsic patterns of the object results which can be captured by a resolution‐constrained microscope. (b) Multiple angles and phases of illumination and capture are obtained to produce complete coverage of the sample. By processing the captured interference patterns with knowledge of the illumination patterns, the unknown structure of the sample can be reconstructed at resolution beyond the conventional band limit of the imaging system. (c) Schematic arrangement of a SIM system. A grating produces a diffraction output that is projected onto the sample as a 3D grid pattern, and the fluorescence emission is captured in a camera‐based wide‐field epi‐fluorescence configuration. (Reprinted with permission from Ref . Copyright 2008 Elsevier). (d) A comparison of confocal and SIM imaging of a mammalian cell nucleus—note the resolution and contrast gain of imaging of the ∼130‐nm nuclear pore complexes. (b and d: Reprinted with permission from Ref . Copyright 2008 AAAS)
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