Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).
TL;DR: A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores that can, in principle, reach molecular-scale resolution is developed.
Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
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TL;DR: This Primer explains the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM, and demonstrates how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology.
1,056 citations
Cites background or methods from "Sub-diffraction-limit imaging by st..."
...This is the idea behind a recently developed super-resolution imaging method called STORM (Rust et al., 2006), PALM (Betzig et al....
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...This is the idea behind a recently developed super-resolution imaging method called STORM (Rust et al., 2006), PALM (Betzig et al., 2006), or FPALM (Hess et al., 2006)....
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...…allows the locations of many fluorophores to be mapped and a super-resolution image constructed from these localizations (Figure 3A), either with synchronized activation (Betzig et al., 2006; Hess et al., 2006; Rust et al., 2006) or with asynchronous activation (Egner et al., 2007)....
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...Using this approach, a lateral image resolution as high as 20 nm has been achieved (Rust et al., 2006)....
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...This second class has been termed stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006), photoactivated localization microscopy (PALM) (Betzig et al....
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TL;DR: Surprisingly, while actin in dendrites formed long filaments, the act in axons was organized into evenly spaced ringlike structures at the axon circumference that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ~180 to 190 nanometers.
Abstract: Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ~180 to 190 nanometers. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin–based cytoskeleton.
1,050 citations
TL;DR: The principles of these methods together with their differences in implementation and operation are discussed, and potential developments are outlined.
Abstract: Until not very long ago, it was widely accepted that lens-based (far-field) optical microscopes cannot visualize details much finer than about half the wavelength of light. The advent of viable physical concepts for overcoming the limiting role of diffraction in the early 1990s set off a quest that has led to readily applicable and widely accessible fluorescence microscopes with nanoscale spatial resolution. Here I discuss the principles of these methods together with their differences in implementation and operation. Finally, I outline potential developments.
978 citations
TL;DR: This work presents an approach for obtaining subdiffraction limit optical resolution in all three dimensions, and demonstrates a 5-fold improvement in spatial resolution by using a conventional wide-field microscope.
Abstract: Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.
975 citations
Cites methods from "Sub-diffraction-limit imaging by st..."
...Rust MJ, Bates M, Zhuang XW (2006) Subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)....
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...Huang B, Jones SA, Brandenburg B, Zhuang XW (2008) Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution....
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...Stochastic techniques using photoswitchable probes have also been developed such as photo-activated localization microscopy (fPALM) (7), stochastic optical reconstruction microscopy (STORM) (8), PALM (9), and variants thereof (10, 11)....
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...Conversely PALM and STORM achieve nanometer resolution, but with the tradeoff of slow acquisition speed (minutes to hours)....
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TL;DR: The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.
Abstract: A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the solution. Because the flux of probes at the object is essentially constant over long time periods, the examination of an almost unlimited number of individual probe molecules becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resolution of ≈25 nm is readily achieved. The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.
943 citations
References
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TL;DR: Multiphoton microscopy has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals and its use is now increasing exponentially.
Abstract: Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.
3,738 citations
TL;DR: A localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations), and results show good agreement with the derived precision equation.
2,390 citations
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
TL;DR: The results strongly support a hand-over-hand model of motility, not an inchworm model, which moves processively on actin.
Abstract: Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of 1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 2x nm and 37 – 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model. Myosin V is a cargo-carrying processive motor
1,888 citations
TL;DR: A family of concepts has emerged that overcomes the diffraction barrier altogether and, relying on saturated optical transitions, these concepts are limited only by the attainable saturation level.
Abstract: For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever
983 citations