This chapter discusses the fundamentals of stimulated emission depletion sted microscopy and aspects of its practical implementation. Osa breaking the diffraction resolution limit by stimulated. Laser scanning confocal microscope is a far field microscopy technique that can provide optically sectioned image can image either with reflected or fluorescence light useful for 3d visualisation of the sample resolution is limited by the diffraction effects stimulated emission depletion. Osa evaluating image resolution in stimulated emission. Feb 26, 2019 stimulated emission depletion sted microscopy is a superresolution fluorescence microscopy technique which can overcome the diffraction limit. Stimulated emission depletion microscopy was the earliest farfield superresolution technique developed hell and wichmann, 1994. Superresolution microscopy an overview sciencedirect. Stimulated emission depletion sted microscopy in research. An indirectly stimulated emission depletion method for. Mar 30, 2018 stimulated emission depletion sted microscopy is a useful tool in investigation for super. Superresolution deep imaging with hollow bessel beam sted.
Sted microscopy utilizes a donutshaped depletion beam to limit the probe volume to be much smaller than a. In particular, stimulated emission depletion sted nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for superresolved imaging in complex biological samples including live organisms 9,10. Precise knowledge of the effective spatial resolution in a stimulated emission depletion sted microscopy experiment is essential for reliable interpretation of the imaging results. Abstractstimulated emission depletion sted microscopy has become a powerful imaging and localized excitation method breaking the diffraction barrier for improved lateral spatial resolution in cellular imaging, lithography, etc. It overcomes the spatial resolution limit imposed by the di. Stimulated emission depletion microscopy physics britannica. Sted is based on nonlinear optical effects that typically require the application of multiple highintensity pulsed lasers with specialized modulation filters to control the excitation beam geometry a technique commonly termed pointspread function engineering. Stimulated emission depletion sted microscopy is one of the most influential nanoscopy techniques. Sted microscopy theoretically provides molecular resolution, but practically different factors limit its resolution. Aug 23, 2018 please use one of the following formats to cite this article in your essay, paper or report. Setup and principal illustration of stimulated emission depletion sted microscopy.
Immunofluorescence stimulated emission depletion microscopy. Aberrations in stimulated emission depletion sted microscopy. The laser beams are moved over the specimen, and an image is gradually built up. In theory, this method allows the spot to be sharpened without limit, but in practice. Ultrafast stimulated emission microscopy of single. Stimulated emission depletion microscopy sted thermo. Stochastic optical reconstruction microscopy storm and stimulated emission depletion sted microscopy are two super. Based on the idea of compressing point spread function psf, a donutshaped depletion beam is overlapped with the excitation beam. In contrast to nearfield scanning optical microscopy, this method can produce threedimensional images of translucent specimens. Stimulated emission depletion microscopy surpasses diffractionlimited microscopy by offering much more information about structures. Stimulated emission depletion sted microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. Pdf we demonstrate stimulated emission depletion sted microscopy implemented in a laser scanning confocal microscope using excitation light derived. The primary modes of superresolution microscopy are stimulated emission depletion microscopy sted, single molecule localization techniques groundstate depletion microscopy gsd, stochastic optical reconstruction microscopy storm, photoactivated localization microscopy palm, and structured illumination microscopy sim.
Sted uses a differential method of two different diffraction patterns, where one pattern excites and the second pattern deexcites fluorochromes. Here, stefan hell, who invented many of these techniques, gives an introduction to these superresolution microscopy techniques, and a detailed discussion of two such techniques. Like all methods of superresolution microscopy, stimulated emission depletion sted microscopy can suffer from the effects of aberrations. One of the most popular superresolution microscopies that breaks the diffraction barrier is stimulated emission depletion sted microscopy.
Stimulated emission depletion sted microscopy improves image resolution by encoding additional spatial information in a second stimulated decay channel with a spatiallyvarying strength. Stimulated emission depletion sted microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of. Axial superresolution by mirrorreflected stimulated. While sted microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy exm provides. An indirectly stimulated emission depletion method for sted. Apr 20, 2011 we demonstrate the first, to our knowledge, integration of stimulated emission depletion sted with selective plane illumination microscopy spim.
It describes important strategies for generating the. Hell and jan wichmann department of medical physics, university of turku, tykistbkatu 6, 20521 turku, finland received march 7, 1994 we propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. Combining afm with superresolution sted stimulated. While sted microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy exm provides subdiffraction resolution by physically enlarging the. Stimulated emission depletion sted microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity. As the optical setup of sted microscopy is based on a laser scanning microscopy lsm system, it potentially has several merits of lsm like confocal or twophoton excitation lsm. Breaking the diffraction resolution limit stimulatedemission.
We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation pointspread function. Stimulated emission depletion sted microscopy, which is typically implemented in a laser scanning confocal microscope system, is the first theoretically proposed and experimentally realized srm technique. Sted microscopy is a process that provides super resolution imaging by selectively. The resolution enhancement is essentially based on switching off the fluorescence of dye molecules by stimulated emission using intense laser light in the outer regions of the. Sted microscopy products, containing high quality fluorescent secondary antibodies and a complete and optimized kit for sample preparation, will provide the best images in high resolution fluorescent sted stimulated emission depletion microscopy. Amplified expansion stimulated emission depletion microscopy. Plasmonic nanoprobes for stimulated emission depletion. Pdf stimulated emission depletion microscopy with a. Because these factors are related to both the sample and the system, a reliable estimation of. This article present a novel implementation of stimulated emission depletion sted microscopy with array detection and photon reassignment, and we make an integrated 19channel array detection and photon reassignment module for convenient engineering applications. Expansion stimulated emission depletion microscopy exsted. Sted stimulated emission depletion and resolft reversible saturable optical fluorescence transitions. Oct 19, 2003 stimulated emission depletion sted microscopy, which relies on saturated depletion of excited molecules at the periphery of a scanning focal spot, has fundamentally overcome the diffraction. Recent research on stimulated emission depletion microscopy.
Stimulated emission depletion sted microscopy fundamentals superresolution microscopy using stimulated emission depletion sted creates subdiffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center. This requires the development of new imaging techniques, both optical and nonoptical. Pdf in recent years, various methods have been proposed to increase the resolution of fluorescence microscopy beyond the diffraction limit of. Stimulated emission depletion sted microscopy is one of the techniques that make up superresolution microscopy. However, the higher is the dose of stimulating photons, the stronger are the photobleaching and phototoxicity effects, which potentially compromise livecell and longterm. Combining sted with timetagged data acquisition methods makes even more information available. It creates superresolution images by the selective deactivation of fluorophores, minimising the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system.
Stimulated emission is the natural counterpart of spontaneous emission, and hence an excited molecule cannot. The instrumentation is similar to a confocal microscope with the addition of a depletion laser, which stimulates excited molecules back to the ground state in a donutshaped region around the. Photonseparation to enhance the spatial resolution of. Stimulated emission depletion sted microscopy, which relies on saturated depletion of excited molecules at the periphery of a scanning focal spot, has fundamentally overcome the diffraction barrier in farfield fluorescence imaging 1,2. Synergic combination of stimulated emission depletion. By silencing the peripheral fluorophores of the excited spot, leaving only the very centre zone vigorous for fluorescence, the effective point spread function psf could be immensely squeezed and subcellular structures, such as organelles, become discernable. Plasmonic nanoprobes for stimulated emission depletion microscopy.
Exploiting the tunability of stimulated emission depletion microscopy. Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. Stimulated emission depletion sted microscopy is a form of scanning far eld uorescence microscop. Stimulated emission depletion microscopy sted picoquant. Stimulated emission depletion sted is one of the most influential nanoscopy or diffraction unlimited microscopy techniques 1,2. As stimulated emission depletion sted microscopy can provide structural details of cells with an optical resolution beyond the diffraction limit, it has become an indispensable tool in cell biology. Stimulated emission depletion microscopy with array detection.
Stimulated emission depletion microscopy sted is a method to resolve structures below the limits of optical resolution and is therefore attributed to superresolution. Sted microscopysuperresolution bioimaging utilizing a. For instance, stimulated emission depletion sted and saturated structured illumination microscopy ssim breach both the assumption of a linear response of a fluorophore to the excitation light and that of a uniform illumination field 17, 18. Stimulated emission depletion sted microscopy literature references. The general optical setup of a sted microscope is based on that of a laser scanning microscope. However, it has since then developed into an observation technology applicable as a flexible turnkey approach in everyday biological research. Stimulated emission depletion sted microscopy, which relies on saturated depletion of excited molecules at the periphery of a scanning focal spot, has fundamentally overcome the diffraction. By breaking the diffraction barrier and providing subdiffraction resolution, stimulated emission depletion sted microscopy 4, 5 has altered longstanding notions about the resolving power of light microscopy and, thus, initiated a hot topic of research at. However, the intense sted laser beam usually causes rapid photobleaching of. Sted microscopy made easy features dec 2014 biophotonics. Jan 29, 2018 stimulated emission depletion sted microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity.
With fluorescence microscopy, cellular structures and proteins of interest can be imaged with very high detail. Hell was awarded the 2014 nobel prize in chemistry for its development. In hells techniquecalled stimulated emission depletion sted microscopyone laser beam excites the fluorescent molecules, but another turns off the fluorescence except from a small area. Zeiss microscopy online campus interactive tutorials sted. Stimulated emission depletion sted microscopy is an advanced spatial superresolution technique and has been successfully commercialized 1, 2. Stimulated emission depletion sted microscopy evolved as the first of such superresolution approaches, but has long been considered as a very complex technique. Stimulated emission depletion microscopy with array. However, sophisticated microscopy architectures and high illumination intensities have limited sted microscopys widespread use. We developed a new class of twophoton excitation stimulated emission depletion 2pested optical microscope. Review of superresolution fluorescence microscopy for. Similarly, it may be possible to track the trajectory of a single virus particle with nanometer precision but still its inner structure remains elusive to light microscopy.
In this work, we show the opportunity to perform superresolved fluorescence imaging, exciting and stimulating the emission of a fluorophore by means of a single wavelength. Stimulated emission depletion sted microscopy is a powerful super resolution microscopy technique that has achieved a series of significant results in breaking the resolution limit and relevant. It creates superresolution images by the selective deactivation of fluorophores, minimising the area of illumination at the focal point, and thus. Ultralow power demand in fluorescence nanoscopy with. We present an approach to threedimensional diffractionunlimited resolution by observing the sample at two optical angles. Stimulated emission depletion microscopy sted is a fluorescence microscopy technique that overcomes the diffraction limited resolution of confocal microscopes. According to albert einstein, when more atoms occupy a higher energy state than a lower one under normal temperature equilibrium see population inversion, it is possible to force atoms to return. Stimulated emission depletion sted microscopy is one type of farfield optical technique demonstrated to provide subdiffraction resolution. Due to specimeninduced aberrations and scattering distortion, it is a great. Breaking the diffraction resolution limit by stimulated. Stimulated emission depletion sted microscopy is one type of farfield optical technique. A spatially shaped doughnut sted beam created by using a phase plate is collinearly combined with an excitation beam.
The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. This seminar presents the basic principles of the technique with the focus on uorescence, stimulated emission, excitation and sted beam, setup of a sted microscope and modi ed resolution equation. Evaluating image resolution in stimulated emission depletion. Combining afm with superresolution sted stimulated emission depletion microscopy research in life sciences demands more and more on the analysis of biological samples with the highest resolution. Abstract expansion microscopy exm enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of.
It has realized a resolution of 20nm in imaging immunelabeled nuclear pore complexes of cultured xenopus cells and a resolution of 5. Sep 23, 2008 by breaking the diffraction barrier and providing subdiffraction resolution, stimulated emission depletion sted microscopy 4, 5 has altered longstanding notions about the resolving power of light microscopy and, thus, initiated a hot topic of research at the interface of physics, chemistry, and biology. Stimulated emission depletion microscopy request pdf. Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging. Jul 29, 2015 stochastic optical reconstruction microscopy storm and stimulated emission depletion sted microscopy are two super. Stimulated emission depletion sted microscopy was first theoretically described by hell and wichmann and, 6 years later, was actually demonstrated to visualize biological nanostructures. Challenges in medical optical imaging fluorescence microscopy is a tool with superb sensitivity that allows individuallabelled biomolecules to be detected by the absorption of light and the reemission of. Nov 17, 20 here, stefan hell, who invented many of these techniques, gives an introduction to these superresolution microscopy techniques, and a detailed discussion of two such techniques. Special issue on stimulated emission depletion microscopy. Zeiss microscopy online campus interactive tutorials. Stimulated emission depletion sted microscopy is a superresolution fluorescence microscopy technique which can overcome the diffraction limit. Stochastic optical reconstruction microscopy storm in.
The resolution limitation is ultimately rooted in the phenomenon of diffraction abbe, 1873. The concept of stimulated emission depletion sted microscopy was first introduced by stefan hell in 1994 and demonstrated that the diffraction barrier could. Stimulated emission depletion microscopy or sted microscopy is a process that provides super resolution imaging figure 1, developed by hell and wichmann, 1994 and demonstrated by hell and klar, 1999. Superresolution microscopy using stimulated emission depletion sted creates subdiffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center of excitation. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells. Find molecular probes fluorescence labels for simulated emission depletion sted imaging, useful to obtain highresolution images in a large depth of field. Review of recent developments in stimulated emission depletion.
Stimulated emission, in laser action, the release of energy from an excited atom by artificial means. The most important aspect of a sted microscope is that the depletion focus maintains a minimum, ideally zero, intensity point that is surrounded by a region of higher intensity. Stimulated emission depletion microscopy sted thermo fisher scientific cn. Stimulated emission depletion sted microscopy is a scanning far eld uorescent microscopy technique with sub wavelength resolution.
In stimulated emission depletion sted microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. Abstract stimulated emission depletion sted microscopy has become a powerful imaging and localized excitation method breaking the diffraction barrier for improved lateral spatial resolution in cellular imaging, lithography, etc. Stimulated emission depletion sted microscopy is routinely used to resolve the ultrastructure of cells with a. It has realized a resolution of 20nm in imaging immunelabeled nuclear pore complexes of cultured xenopus cells and a. The resolution enhancement is essentially based on switching off the fluorescence of dye molecules by stimulated emission using intense laser light in the outer regions of the diffraction limited excitation focus. Stimulated emission depletion microscopy sted is one of the pivotal superresolution techniques.