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.
Citations
More filters
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
TL;DR: 3D stochastic optical reconstruction microscopy (STORM) is demonstrated by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy, allowing the 3D morphology of nanoscopic cellular structures to be resolved.
Abstract: 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.
2,589 citations
[...]
TL;DR: Optical antennas are devices that convert freely propagating optical radiation into localized energy, and vice versa as mentioned in this paper, and hold promise for enhancing the performance and efficiency of photodetection, light emission and sensing.
Abstract: Optical antennas are devices that convert freely propagating optical radiation into localized energy, and vice versa. They enable the control and manipulation of optical fields at the nanometre scale, and hold promise for enhancing the performance and efficiency of photodetection, light emission and sensing. Although many of the properties and parameters of optical antennas are similar to their radiowave and microwave counterparts, they have important differences resulting from their small size and the resonant properties of metal nanostructures. This Review summarizes the physical properties of optical antennas, provides a summary of some of the most important recent developments in the field, discusses the potential applications and identifies the future challenges and opportunities.
2,557 citations
TL;DR: A practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes, is provided.
Abstract: Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.
1,929 citations
Cites background from "Sub-diffraction-limit imaging by st..."
...28 ), a side effect that has been exploited for super-resolution photoswitching imaging application...
[...]
TL;DR: Switches, and Actuators Masahiro Irie,*, Tuyoshi Fukaminato,‡ Kenji Matsuda, and Seiya Kobatake.
Abstract: Switches, and Actuators Masahiro Irie,*,† Tuyoshi Fukaminato,‡ Kenji Matsuda, and Seiya Kobatake †Research Center for Smart Molecules, Rikkyo University, Nishi-Ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan ‡Research Institute for Electronic Science, Hokkaido University, N20, W10, Kita-ku, Sapporo 001-0020, Japan Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan Department of Applied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan
1,884 citations
References
More filters
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