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Journal ArticleDOI

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.

01 May 2000-Journal of Microscopy (Blackwell Science Ltd)-Vol. 198, Iss: 2, pp 82-87
TL;DR: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide‐field fluorescence microscope with strikingly increased clarity compared to both conventional and confocal microscopes.
Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.

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Citations
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Journal ArticleDOI
15 Sep 2006-Science
TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Abstract: We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

7,924 citations

Journal ArticleDOI
24 May 2012-Neuron
TL;DR: It is shown that microglia engulf presynaptic inputs during peak retinogeniculate pruning and that engulfment is dependent upon neural activity and themicroglia-specific phagocytic signaling pathway, complement receptor 3(CR3)/C3.

2,864 citations


Cites methods from "Surpassing the lateral resolution l..."

  • ...3D structural illuminationmicroscopy (3D-SIM), a technique enabling 2X the resolution of light microscopy (Gustafsson, 2000), was used to assess the P5 dLGN of CX3CR1+/EGFP mice immunolabeled for VGlut2....

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Journal ArticleDOI
25 May 2007-Science
TL;DR: 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.
Abstract: 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.

2,730 citations

Journal ArticleDOI
TL;DR: Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples, and a recently proposed method in which the nonlinearity arises from saturation of the excited state is experimentally demonstrated.
Abstract: 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 arisesfromsaturationoftheexcitedstate.Thismethodcanbeused 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.

2,125 citations

Journal ArticleDOI
TL;DR: It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.
Abstract: Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

1,534 citations


Cites background from "Surpassing the lateral resolution l..."

  • ...Periodic illumination patterns can be created through the interference of multiple light sources in the axial direction (18), the lateral direction (19), or both (20)....

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References
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BookDOI
01 Jan 1990
TL;DR: Methods for Three-Dimensional Imaging and Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen.
Abstract: Foundations of Confocal Scanned Imaging in Light Microscopy -- Fundamental Limits in Confocal Microscopy -- Special Optical Elements -- Points, Pixels, and Gray Levels: Digitizing Image Data -- Laser Sources for Confocal Microscopy -- Non-Laser Light Sources for Three-Dimensional Microscopy -- Objective Lenses for Confocal Microscopy -- The Contrast Formation in Optical Microscopy -- The Intermediate Optical System of Laser-Scanning Confocal Microscopes -- Disk-Scanning Confocal Microscopy -- Measuring the Real Point Spread Function of High Numerical Aperture Microscope Objective Lenses -- Photon Detectors for Confocal Microscopy -- Structured Illumination Methods -- Visualization Systems for Multi-Dimensional Microscopy Images -- Automated Three-Dimensional Image Analysis Methods for Confocal Microscopy -- Fluorophores for Confocal Microscopy: Photophysics and Photochemistry -- Practical Considerations in the Selection and Application of Fluorescent Probes -- Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy -- Confocal Microscopy of Living Cells -- Aberrations in Confocal and Multi-Photon Fluorescence Microscopy Induced by Refractive Index Mismatch -- Interaction of Light with Botanical Specimens -- Signal-to-Noise Ratio in Confocal Microscopes -- Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging -- Blind Deconvolution -- Image Enhancement by Deconvolution -- Fiber-Optics in Scanning Optical Microscopy -- Fluorescence Lifetime Imaging in Scanning Microscopy -- Multi-Photon Molecular Excitation in Laser-Scanning Microscopy -- Multifocal Multi-Photon Microscopy -- 4Pi Microscopy -- Nanoscale Resolution with Focused Light: Stimulated Emission Depletion and Other Reversible Saturable Optical Fluorescence Transitions Microscopy Concepts -- Mass Storage, Display, and Hard Copy -- Coherent Anti-Stokes Raman Scattering Microscopy -- Related Methods for Three-Dimensional Imaging -- Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen -- Practical Confocal Microscopy -- Selective Plane Illumination Microscopy -- Cell Damage During Multi-Photon Microscopy -- Photobleaching -- Nonlinear (Harmonic Generation) Optical Microscopy -- Imaging Brain Slices -- Fluorescent Ion Measurement -- Confocal and Multi-Photon Imaging of Living Embryos -- Imaging Plant Cells -- Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells -- Automated Confocal Imaging and High-Content Screening for Cytomics -- Automated Interpretation of Subcellular Location Patterns from Three-Dimensional Confocal Microscopy -- Display and Presentation Software -- When Light Microscope Resolution Is Not Enough:Correlational Light Microscopy and Electron Microscopy -- Databases for Two- and Three-Dimensional Microscopical Images in Biology -- Confocal Microscopy of Biofilms — Spatiotemporal Approaches -- Bibliography of Confocal Microscopy.

4,121 citations


"Surpassing the lateral resolution l..." refers background in this paper

  • ...Although some recent non-linear concepts show promise (Klar & Hell, 1999), only one established technique has the ability to go beyond the limit in principle, namely confocal fluorescence microscopey (Minsky, 1961; Pawley, 1995)....

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Journal ArticleDOI
01 Dec 1873

2,157 citations

Journal ArticleDOI
TL;DR: A simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object and processing images that are substantially similar to those obtained with confocal microscopes is described.
Abstract: We describe a simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object. Images taken at three spatial positions of the grid are processed in real time to produce optically sectioned images that are substantially similar to those obtained with confocal microscopes.

1,138 citations


"Surpassing the lateral resolution l..." refers background or methods in this paper

  • ...In fact, the resolution performance of wide-field microscopes in the third dimension can itself be enhanced by laterally structured illumination similar to that used here (Neil et al., 1997; Neil et al., 1998)....

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  • ...The last approach can enhance the axial sectioning ability further by making the illumination vary axially as well as laterally 2 this effect is similar to that of illuminating the coarser grating with fully incoherent light (Neil et al., 1997), but maintains the maximum lateral resolution enhancement and strong pattern contrast....

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  • ...…axial sectioning ability further by making the illumination vary axially as well as laterally 2 this effect is similar to that of illuminating the coarser grating with fully incoherent light (Neil et al., 1997), but maintains the maximum lateral resolution enhancement and strong pattern contrast....

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Journal ArticleDOI
TL;DR: The resolution limit of scanning far-field fluorescence microscopy is overcame by disabling the fluorescence from the outer part of the focal spot by a spatially offset pulse.
Abstract: We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150±8 nm to 106±8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.

789 citations


"Surpassing the lateral resolution l..." refers background in this paper

  • ...Although some recent non-linear concepts show promise (Klar & Hell, 1999), only one established technique has the ability to go beyond the limit in principle, namely confocal fluorescence microscopey (Minsky, 1961; Pawley, 1995)....

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Book ChapterDOI
TL;DR: This chapter has discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information and developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen.
Abstract: The combination of the specificity provided by fluorescence microscopy and the ability to quantitatively analyze specimens in three dimensions allows the fundamental organization of cells to be probed as never before Key features in this emergent technology have been the development of a wide variety of fluorescent dyes or fluorescently labeled probes to provide the requisite specificity High-quality, cooled charge-coupled devices have recently become available Functioning as nearly ideal imagers or "electronic film," they are more sensitive than photomultipliers and provide extraordinarily accurate direct digital readout from the microscope Not only is this precision crucial for accurate quantitative imaging such as that required for the ratioing necessary to determine intracellular ion concentrations, but it also opens the way for sophisticated image processing It is important to realize that image processing isn't simply a means to improve image aesthetics, but can directly provide new, biologically important information The impact of modern video microscopy techniques (Allen, 1985; Inoue, 1986) attests to the fact that many biologically relevant phenomena take place at the limits of conventional microscopy Image processing can be used to substantially enhance the resolution and contrast obtainable in two dimensions, enabling the invisible to be seen and quantitated Cells are intrinsically three-dimensional This can simply be a nuisance because of limited depth of focus of the microscope or it could be a fundamental aspect of the problem being studied In either case, image processing techniques can be used to rapidly provide the desired representation of the data In this chapter we have discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information The most straightforward of these methods uses only information from adjacent focal planes to correct the central one This approach can be readily applied to virtually any problem and with most commonly available image processing hardware to provide a substantially deblurred image in almost real time In addition to covering more sophisticated algorithms where the utmost in three-dimensional imaging is required, we have developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen This is equivalent to that produced by a microscope having the impossible combination of a high-NA objective lens and an infinite depth of focus A variation on this method allows efficient calculation of stereo pairs(ABSTRACT TRUNCATED AT 400 WORDS)

702 citations


"Surpassing the lateral resolution l..." refers background in this paper

  • ...It is known that non-linear, constrained deconvolution algorithms can enhance apparent resolution by making use of a priori information about the sample, such as the fact that the dye density is non-negative (Agard et al., 1989; Carrington et al., 1995)....

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