STED microscopy

Last updated
Stimulated emission depletion (STED) microscopy provides significant resolution improvements over those possible with Confocal microscopy. STED Confocal Comparison 50nm HWFM.png
Stimulated emission depletion (STED) microscopy provides significant resolution improvements over those possible with Confocal microscopy.

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. [1] It was developed by Stefan W. Hell and Jan Wichmann in 1994, [2] and was first experimentally demonstrated by Hell and Thomas Klar in 1999. [3] Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin [4] (Institute of Biophysics, USSR Academy of Sciences, Siberian Branch, Krasnoyarsk) had patented the STED idea. [5] This patent was unknown to Hell and Wichmann in 1994.

Contents

STED microscopy is one of several types of super resolution microscopy techniques that have recently been developed to bypass the diffraction limit of light microscopy to increase resolution. STED is a deterministic functional technique that exploits the non-linear response of fluorophores commonly used to label biological samples in order to achieve an improvement in resolution, that is to say STED allows for images to be taken at resolutions below the diffraction limit. This differs from the stochastic functional techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) as these methods use mathematical models to reconstruct a sub diffraction limit from many sets of diffraction limited images.

Background

Ernst Abbe's formula for the diffraction limit, set in stone at a monument in Jena. Ernst-Abbe-Denkmal Jena Furstengraben - 20140802 125708.jpg
Ernst Abbe's formula for the diffraction limit, set in stone at a monument in Jena.
Jablonski diagram showing the redshift of the stimulated photon. This redshift allows the stimulated photon to be ignored. STED Jablonski.jpg
Jablonski diagram showing the redshift of the stimulated photon. This redshift allows the stimulated photon to be ignored.
Diagram of the design of a STED device. The double laser design allows for excitation and stimulated emission to be used together for STED. STED Insturmentation.jpg
Diagram of the design of a STED device. The double laser design allows for excitation and stimulated emission to be used together for STED.

In traditional microscopy, the resolution that can be obtained is limited by the diffraction of light. Ernst Abbe developed an equation to describe this limit. The equation is:

where D is the diffraction limit, λ is the wavelength of the light, and NA is the numerical aperture, or the refractive index of the medium multiplied by the sine of the angle of incidence. n describes the refractive index of the specimen, α measures the solid half‐angle from which light is gathered by an objective, λ is the wavelength of light used to excite the specimen, and NA is the numerical aperture. To obtain high resolution (i.e. small d values), short wavelengths and high NA values (NA = n sinα) are optimal. [6] This diffraction limit is the standard by which all super resolution methods are measured. Because STED selectively deactivates the fluorescence, it can achieve resolution better than traditional confocal microscopy. Normal fluorescence occurs by exciting an electron from the ground state into an excited electronic state of a different fundamental energy level (S0 goes to S1) which, after relaxing back to the ground state (of S1), emits a photon by dropping from S1 to a vibrational energy level on S0. STED interrupts this process before the photon is released. The excited electron is forced to relax into a higher vibration state than the fluorescence transition would enter, causing the photon to be released to be red-shifted as shown in the image to the right. [7] Because the electron is going to a higher vibrational state, the energy difference of the two states is lower than the normal fluorescence difference. This lowering of energy raises the wavelength, and causes the photon to be shifted farther into the red end of the spectrum. This shift differentiates the two types of photons, and allows the stimulated photon to be ignored.

To force this alternative emission to occur, an incident photon must strike the fluorophore. This need to be struck by an incident photon has two implications for STED. First, the number of incident photons directly impacts the efficiency of this emission, and, secondly, with sufficiently large numbers of photons fluorescence can be completely suppressed. [8] To achieve the large number of incident photons needed to suppress fluorescence, the laser used to generate the photons must be of a high intensity. Unfortunately, this high intensity laser can lead to the issue of photobleaching the fluorophore. Photobleaching is the name for the destruction of fluorophores by high intensity light.

Process

Comparison of confocal microscopy and STED microscopy. This shows the improved resolution of STED microscopy over traditional techniques. 2color-STED-example.png
Comparison of confocal microscopy and STED microscopy. This shows the improved resolution of STED microscopy over traditional techniques.
Excitation spot (2D, left), doughnut-shape de-excitation spot (center) and remaining area allowing fluorescence (right). STED Mikroskop PSFs.jpg
Excitation spot (2D, left), doughnut-shape de-excitation spot (center) and remaining area allowing fluorescence (right).

STED functions by depleting fluorescence in specific regions of the sample while leaving a center focal spot active to emit fluorescence. This focal area can be engineered by altering the properties of the pupil plane of the objective lens. [9] [10] [11] The most common early example of these diffractive optical elements, or DOEs, is a torus shape used in two-dimensional lateral confinement shown below. The red zone is depleted, while the green spot is left active. This DOE is generated by a circular polarization of the depletion laser, combined with an optical vortex. The lateral resolution of this DOE is typically between 30 and 80 nm. However, values down to 2.4 nm have been reported. [12] Using different DOEs, axial resolution on the order of 100 nm has been demonstrated. [13] A modified Abbe's equation describes this sub diffraction resolution as:

Where is the refractive index of the medium, is the intracavity intensity and is the saturation intensity. Where is the saturation factor expressing the ratio of the applied (maximum) STED intensity to the saturation intensity, . [6] [14]

To optimize the effectiveness of STED, the destructive interference in the center of the focal spot needs to be as close to perfect as possible. That imposes certain constraints on the optics that can be used.

Dyes

Early on in the development of STED, the number of dyes that could be used in the process was very limited. Rhodamine B was named in the first theoretical description of STED. [2] As a result, the first dyes used were laser emitting in the red spectrum. To allow for STED analysis of biological systems, the dyes and laser sources must be tailored to the system. This desire for better analysis of these systems has led to living cell STED and multicolor STED, but it has also demanded more and more advanced dyes and excitation systems to accommodate the increased functionality. [7]

One such advancement was the development of immunolabeled cells. These cells are STED fluorescent dyes bound to antibodies through amide bonds. The first use of this technique coupled MR-121SE, a red dye, with a secondary anti-mouse antibody. [8] Since that first application, this technique has been applied to a much wider range of dyes including green emitting, Atto 532, [15] [16] [17] and yellow emitting, Atto 590, [18] as well as additional red emitting dyes. In addition, Atto 647N was first used with this method to produce two-color STED. [19]

Applications

Over the last several years, STED has developed from a complex and highly specific technique to a general fluorescence method. As a result, a number of methods have been developed to expand the utility of STED and to allow more information to be provided.

Structural analysis

From the beginning of the process, STED has allowed fluorescence microscopy to perform tasks that had been only possible using electron microscopy. As an example, STED was used for the elucidation of protein structure analysis at a sub-organelle level. The common proof of this level of study is the observation of cytoskeletal filaments. In addition, neurofilaments, actin, and tubulin are often used to compare the resolving power of STED and confocal microscopes. [20] [21] [22]

Using STED, a lateral resolution of 70 – 90 nm has been achieved while examining SNAP25, a human protein that regulates membrane fusion. This observation has shown that SNAP25 forms clusters independently of the SNARE motif's functionality, and binds to clustered syntaxin. [23] [24] Studies of complex organelles, like mitochondria, also benefit from STED microscopy for structural analysis. Using custom-made STED microscopes with a lateral resolution of fewer than 50 nm, mitochondrial proteins Tom20, VDAC1, and COX2 were found to distribute as nanoscale clusters. [25] [26] Another study used a homemade STED microscopy and DNA binding fluorescent dye, measured lengths of DNA fragments much more precisely than conventional measurement with confocal microscopy. [27]

Correlative methods

Due to its function, STED microscopy can often be used with other high-resolution methods. The resolution of both electron and atomic force microscopy is even better than STED resolution, but by combining atomic force with STED, Shima et al. were able to visualize the actin cytoskeleton of human ovarian cancer cells while observing changes in cell stiffness. [28]

Multicolor

Multicolor STED was developed in response to a growing problem in using STED to study the dependency between structure and function in proteins. To study this type of complex system, at least two separate fluorophores must be used. Using two fluorescent dyes and beam pairs, colocalized imaging of synaptic and mitochondrial protein clusters is possible with a resolution down to 5 nm [18]. Multicolor STED has also been used to show that different populations of synaptic vesicle proteins do not mix of escape synaptic boutons. [29] [30] By using two color STED with multi-lifetime imaging, three channel STED is possible.

Live-cell

Early on, STED was thought to be a useful technique for working with living cells. [13] Unfortunately, the only way for cells to be studied was to label the plasma membrane with organic dyes. [29] Combining STED with fluorescence correlation spectroscopy showed that cholesterol-mediated molecular complexes trap sphingolipids, but only transiently. [31] However, only fluorescent proteins provide the ability to visualize any organelle or protein in a living cell. This method was shown to work at 50 nm lateral resolution within Citrine-tubulin expressing mammalian cells. [32] [33] In addition to detecting structures in mammalian cells, STED has allowed for the visualization of clustering YFP tagged PIN proteins in the plasma membrane of plant cells. [34]

Recently, multicolor live-cell STED was performed using a pulsed far-red laser and CLIPf-tag and SNAPf-tag expression. [35]

In the brain of intact animals

Superficial layers of mouse cortex can be repetitively imaged through a cranial window. [36] This allows following the fate and shape of individual dendritic spines for many weeks. [37] With two-color STED, it is even possible to resolve the nanostructure of the postsynaptic density in life animals. [38]

STED at video rates and beyond

Super-resolution requires small pixels, which means more spaces to acquire from in a given sample, which leads to a longer acquisition time. However, the focal spot size is dependent on the intensity of the laser being used for depletion. As a result, this spot size can be tuned, changing the size and imaging speed. A compromise can then be reached between these two factors for each specific imaging task. Rates of 80 frames per second have been recorded, with focal spots around 60 nm. [1] [39] Up to 200 frames per second can be reached for small fields of view. [40]

Problems

Photobleaching can occur either from excitation into an even higher excited state, or from excitation in the triplet state. To prevent the excitation of an excited electron into another, higher excited state, the energy of the photon needed to trigger the alternative emission should not overlap the energy of the excitation from one excited state to another. [41] This will ensure that each laser photon that contacts the fluorophores will cause stimulated emission, and not cause the electron to be excited to another, higher energy state. Triplet states are much longer lived than singlet states, and to prevent triplet states from exciting, the time between laser pulses needs to be long enough to allow the electron to relax through another quenching method, or a chemical compound should be added to quench the triplet state. [20] [42] [43]

See also

Related Research Articles

<span class="mw-page-title-main">Microscopy</span> Viewing of objects which are too small to be seen with the naked eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

<span class="mw-page-title-main">Optical microscope</span> Microscope that uses visible light

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.

<span class="mw-page-title-main">Immunofluorescence</span> Technique used for light microscopy

Immunofluorescence(IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens. The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

<span class="mw-page-title-main">Fluorescence microscope</span> Optical microscope that uses fluorescence and phosphorescence

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

<span class="mw-page-title-main">Confocal microscopy</span> Optical imaging technique

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

<span class="mw-page-title-main">Two-photon excitation microscopy</span> Fluorescence imaging technique

Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

<span class="mw-page-title-main">Photobleaching</span> Loss of colour by a pigment when illuminated

In optics, photobleaching is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules. Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores. The number of excitation cycles to achieve full bleaching varies. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy.

<span class="mw-page-title-main">Molecular imaging</span> Imaging molecules within living patients

Molecular imaging is a field of medical imaging that focuses on imaging molecules of medical interest within living patients. This is in contrast to conventional methods for obtaining molecular information from preserved tissue samples, such as histology. Molecules of interest may be either ones produced naturally by the body, or synthetic molecules produced in a laboratory and injected into a patient by a doctor. The most common example of molecular imaging used clinically today is to inject a contrast agent into a patient's bloodstream and to use an imaging modality to track its movement in the body. Molecular imaging originated from the field of radiology from a need to better understand fundamental molecular processes inside organisms in a noninvasive manner.

A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy.

RESOLFT, an acronym for REversible Saturable OpticaLFluorescence Transitions, denotes a group of optical fluorescence microscopy techniques with very high resolution. Using standard far field visible light optics a resolution far below the diffraction limit down to molecular scales can be obtained.

<span class="mw-page-title-main">Stefan Hell</span> Romanian-German physicist (born 1962)

Stefan Walter Hell is a Romanian-German physicist and one of the directors of the Max Planck Institute for Multidisciplinary Sciences in Göttingen, and of the Max Planck Institute for Medical Research in Heidelberg, both of which are in Germany. He received the Nobel Prize in Chemistry in 2014 "for the development of super-resolved fluorescence microscopy", together with Eric Betzig and William Moerner.

<span class="mw-page-title-main">GSD microscopy</span> Method of microscopy

Ground state depletion microscopy is an implementation of the RESOLFT concept. The method was proposed in 1995 and experimentally demonstrated in 2007. It is the second concept to overcome the diffraction barrier in far-field optical microscopy published by Stefan Hell. Using nitrogen-vacancy centers in diamonds a resolution of up to 7.8 nm was achieved in 2009. This is far below the diffraction limit (~200 nm).

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.

<span class="mw-page-title-main">Light sheet fluorescence microscopy</span> Fluorescence microscopy technique

Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction. A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because light sheet fluorescence microscopy scans samples by using a plane of light instead of a point, it can acquire images at speeds 100 to 1,000 times faster than those offered by point-scanning methods.

Super-resolution dipole orientation mapping (SDOM) is a form of fluorescence polarization microscopy (FPM) that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization (FP) is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.

Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by , which is much faster than that of two-photon microscopy by . In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three-photon microscopy to have higher resolution than conventional microscopy.

Ilaria Testa is an Italian-born scientist who is a Fellow at the SciLifeLab in Stockholm and an Associate Professor at the Department of Applied Physics at the School of Engineering Science at the KTH Royal Institute of Technology. She has made major contributions to advanced microscopy, particularly superresolution microscopy.

MINFLUX, or minimal fluorescence photon fluxes microscopy, is a super-resolution light microscopy method that images and tracks objects in two and three dimensions with single-digit nanometer resolution.

References

  1. 1 2 Westphal, V.; S. O. Rizzoli; M. A. Lauterbach; D. Kamin; R. Jahn; S. W. Hell (2008). "Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement". Science. 320 (5873): 246–249. Bibcode:2008Sci...320..246W. doi: 10.1126/science.1154228 . PMID   18292304. S2CID   14169050.
  2. 1 2 Hell, S. W.; Wichmann, J. (1994). "Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy". Optics Letters. 19 (11): 780–782. Bibcode:1994OptL...19..780H. doi:10.1364/OL.19.000780. PMID   19844443.
  3. ↑ Klar, Thomas A.; Stefan W. Hell (1999). "Subdiffraction resolution in far-field fluorescence microscopy". Optics Letters. 24 (14): 954–956. Bibcode:1999OptL...24..954K. doi:10.1364/OL.24.000954. PMID   18073907.
  4. ↑ "Victor Okhonin".
  5. ↑ Okhonin V.A., A method of examination of sample microstructure, Patent SU 1374922, (See also in the USSR patents database SU 1374922) priority date April 10, 1986, Published on July 30, 1991, Soviet Patents Abstracts, Section EI, Week 9218, Derwent Publications Ltd., London, GB; Class S03, p. 4. Cited by patents US 5394268 A (1993) and US RE38307 E1 (1995). From the English translation: "The essence of the invention is as follows. Luminescence is excited in a sample placed in the field of several standing light waves, which cause luminescence quenching because of stimulated transitions...".
  6. 1 2 Blom, H.; Brismar, H. (2014). "STED microscopy: Increased resolution for medical research?". Journal of Internal Medicine. 276 (6): 560–578. doi: 10.1111/joim.12278 . PMID   24980774.
  7. 1 2 Müller, T.; Schumann, C.; Kraegeloh, A. (2012). "STED Microscopy and its Applications: New Insights into Cellular Processes on the Nanoscale". ChemPhysChem. 13 (8): 1986–2000. doi:10.1002/cphc.201100986. PMID   22374829.
  8. 1 2 Dyba, M.; Hell, S. W. (2003). "Photostability of a Fluorescent Marker Under Pulsed Excited-State Depletion through Stimulated Emission". Applied Optics. 42 (25): 5123–5129. Bibcode:2003ApOpt..42.5123D. doi:10.1364/AO.42.005123. PMID   12962391.
  9. ↑ Török, P.; Munro, P. R. T. (2004). "The use of Gauss-Laguerre vector beams in STED microscopy". Optics Express. 12 (15): 3605–3617. Bibcode:2004OExpr..12.3605T. doi: 10.1364/OPEX.12.003605 . PMID   19483892.
  10. ↑ Keller, J.; Schönle, A.; Hell, S. W. (2007). "Efficient fluorescence inhibition patterns for RESOLFT microscopy". Optics Express. 15 (6): 3361–3371. Bibcode:2007OExpr..15.3361K. doi: 10.1364/OE.15.003361 . PMID   19532577. S2CID   31855914.
  11. ↑ S. W. Hell, Reuss, M (Jan 2010). "Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation". Optics Express. 18 (2): 1049–58. Bibcode:2010OExpr..18.1049R. doi: 10.1364/OE.18.001049 . PMID   20173926.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  12. ↑ Wildanger, D.; B. R. Patton; H. Schill; L. Marseglia; J. P. Hadden; S. Knauer; A. Schönle; J. G. Rarity; J. L. O’Brien; S. W. Hell; J. M. Smith (2012). "Solid Immersion Facilitates Fluorescence Microscopy with Nanometer Resolution and Sub-Ångström Emitter Localization". Advanced Materials. 24 (44): OP309–OP313. Bibcode:2012AdM....24P.309W. doi:10.1002/adma.201203033. PMC   3546393 . PMID   22968917.
  13. 1 2 Klar, T. A.; S. Jakobs; M. Dyba; A. Egner; S. W. Hell (2000). "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission". Proc. Natl. Acad. Sci. U.S.A. 97 (15): 8206–8210. Bibcode:2000PNAS...97.8206K. doi: 10.1073/pnas.97.15.8206 . PMC   26924 . PMID   10899992.
  14. ↑ Hell, Stefan W. (November 2003). "Toward fluorescence nanoscopy". Nature Biotechnology. 21 (11): 1347–1355. doi:10.1038/nbt895. ISSN   1546-1696. PMID   14595362. S2CID   25695312.
  15. ↑ Lang, Sieber (April 2006). "The SNARE Motif Is Essential for the Formation of Syntaxin Clusters in the Plasma Membrane". Biophysical Journal. 90 (8): 2843–2851. Bibcode:2006BpJ....90.2843S. doi:10.1529/biophysj.105.079574. PMC   1414554 . PMID   16443657.
  16. ↑ Sieber, J. J.; K. L. Willig; R. Heintzmann; S. W. Hell; T. Lang (2006). "The SNARE Motif Is Essential for the Formation of Syntaxin Clusters in the Plasma Membrane". Biophys. J. 90 (8): 2843–2851. Bibcode:2006BpJ....90.2843S. doi:10.1529/biophysj.105.079574. PMC   1414554 . PMID   16443657.
  17. ↑ Willig, K. I.; J. Keller; M. Bossi; S. W. Hell (2006). "STED microscopy resolves nanoparticle assemblies". New J. Phys. 8 (6): 106. Bibcode:2006NJPh....8..106W. doi: 10.1088/1367-2630/8/6/106 .
  18. ↑ Wildanger, D.; Rittweger; Kastrup, L.; Hell, S. W. (2008). "STED microscopy with a supercontinuum laser source". Opt. Express. 16 (13): 9614–9621. Bibcode:2008OExpr..16.9614W. doi: 10.1364/oe.16.009614 . PMID   18575529. S2CID   38016354.
  19. ↑ Doonet, G.; J. Keller; C. A. Wurm; S. O. Rizzoli; V. Westphal; A. Schonle; R. Jahn; S. Jakobs; C. Eggeling; S. W. Hell (2007). "Two-Color Far-Field Fluorescence Nanoscopy". Biophys. J. 92 (8): L67–L69. Bibcode:2007BpJ....92L..67D. doi:10.1529/biophysj.107.104497. PMC   1831704 . PMID   17307826.
  20. 1 2 Kasper, R.; B. Harke; C. Forthmann; P. Tinnefeld; S. W. Hell; M. Sauer (2010). "Single-Molecule STED Microscopy with Photostable Organic Fluorophores". Small. 6 (13): 1379–1384. doi:10.1002/smll.201000203. PMID   20521266.
  21. ↑ Willig, K. I.; B. Harke; R. Medda; S. W. Hell (2007). "STED microscopy with continuous wave beams". Nat. Methods. 4 (11): 915–918. doi:10.1038/nmeth1108. hdl: 11858/00-001M-0000-0012-DEE7-E . PMID   17952088. S2CID   5576096.
  22. ↑ Buckers, J.; D. Wildanger; G. Vicidomini; L. Kastrup; S. W. Hell (2011). "Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses". Opt. Express. 19 (4): 3130–3143. Bibcode:2011OExpr..19.3130B. doi: 10.1364/OE.19.003130 . PMID   21369135. S2CID   38820566.
  23. ↑ Halemani, N. D.; I. Bethani; S. O. Rizzoli; T. Lang (2010). "Structure and Dynamics of a Two-Helix SNARE Complex in Live Cells". Traffic. 11 (3): 394–404. doi: 10.1111/j.1600-0854.2009.01020.x . PMID   20002656. S2CID   22375304.
  24. ↑ Geumann, U.; C. Schäfer; D. Riedel; R. Jahn; S. O. Rizzoli (2010). "Synaptic membrane proteins form stable microdomains in early endosomes". Microsc. Res. Tech. 73 (6): 606–617. doi:10.1002/jemt.20800. PMID   19937745. S2CID   5278558.
  25. ↑ Singh, H.; R. Lu; P. F. G. Rodriguez; Y. Wu; J. C. Bopassa; E. Stefani; L. ToroMitochondrion (2012). "Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy". Mitochondrion. 12 (2): 230–236. doi:10.1016/j.mito.2011.09.004. PMC   3258335 . PMID   21982778.
  26. ↑ Wurm, C. A.; D. Neumann; R. Schmidt; A. Egner; S. Jakobs (2010). "Sample Preparation for STED Microscopy". Live Cell Imaging. Methods in Molecular Biology. Vol. 591. pp. 185–199. doi:10.1007/978-1-60761-404-3_11. hdl:11858/00-001M-0000-0012-D68F-7. ISBN   978-1-60761-403-6. PMID   19957131.
  27. ↑ Kim, Namdoo; Kim, Hyung Jun; Kim, Younggyu; Min, Kyung Suk; Kim, Seong Keun (2016). "Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy". Analytical and Bioanalytical Chemistry. 408 (23): 6453–6459. doi:10.1007/s00216-016-9764-9. PMID   27457103. S2CID   5591747.
  28. ↑ Sharma, S.; C. Santiskulvong; L. Bentolila; J. Rao; O. Dorigo; J. K. Gimzewski (2011). "Correlative nanomechanical profiling with super-resolution F-actin imaging reveals novel insights into mechanisms of cisplatin resistance in ovarian cancer cells". Nanomedicine: Nanotechnology, Biology and Medicine. 8 (5): 757–766. doi:10.1016/j.nano.2011.09.015. PMID   22024198.
  29. 1 2 Hoopman, P.; A. Punge; S. V. Barysch; V. Westphal; J. Buchkers; F. Opazo; I. Bethani; M. A. Lauterbach; S. W. Hell; S. O. Rizzoli (2010). "Endosomal sorting of readily releasable synaptic vesicles" (PDF). Proc. Natl. Acad. Sci. U.S.A. 107 (44): 19055–19060. Bibcode:2010PNAS..10719055H. doi: 10.1073/pnas.1007037107 . PMC   2973917 . PMID   20956291.
  30. ↑ Opazo, F.; A. Punge; J. Buckers; P. Hoopmann; L. Kastrup; S. W. Hell; S. O. Rizzoli (2010). "Limited intermixing of synaptic vesicle components upon vesicle recycling". Traffic. 11 (6): 800–812. doi: 10.1111/j.1600-0854.2010.01058.x . PMID   20230528. S2CID   16847327.
  31. ↑ Eggeling, C.; Ringemann, C.; Medda, R.; Schwarzmann, G.; Sandhoff, K.; Polyakova, S.; Belov, V. N.; Hein, B.; von Middendorff, C.; Schonle, A.; Hell, S. W. (2009). "Direct observation of the nanoscale dynamics of membrane lipids in a living cell". Nature. 457 (7233): 1159–1162. Bibcode:2009Natur.457.1159E. doi:10.1038/nature07596. hdl: 11858/00-001M-0000-0012-D8CA-4 . PMID   19098897. S2CID   4428863.
  32. ↑ Willig, K. I.; R. R. Kellner; R. Medda; B. Heln; S. Jakobs; S. W. Hell (2006). "Nanoscale resolution in GFP-based microscopy". Nat. Methods. 3 (9): 721–723. doi:10.1038/nmeth922. hdl: 11858/00-001M-0000-0012-5CC4-1 . PMID   16896340. S2CID   9887386.
  33. ↑ Hein, B.; K. I. Willig; S. W. Hell (2008). "Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell". Proc. Natl. Acad. Sci. U.S.A. 105 (38): 14271–14276. Bibcode:2008PNAS..10514271H. doi: 10.1073/pnas.0807705105 . PMC   2538451 . PMID   18796604.
  34. ↑ Kleine-Vehn, J.; Wabnik, K.; Martiniere, A.; Langowski, L.; Willig, K.; Naramoto, S.; Leitner, J.; Tanaka, H.; Jakobs, S.; Robert, S.; Luschnig, C.; Govaerts, W.; Hell, S. W.; Runions, J.; Friml, J. (2011). "Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane". Mol. Syst. Biol. 7: 540. doi:10.1038/msb.2011.72. PMC   3261718 . PMID   22027551.
  35. ↑ Pellett, P. A.; X. Sun; T. J. Gould; J. E. Rothman; M. Q. Xu; I. R. Corréa; J. Bewersdorf (2011). "Two-color STED microscopy in living cells". Biomed. Opt. Express. 2 (8): 2364–2371. doi:10.1364/boe.2.002364. PMC   3149534 . PMID   21833373.
  36. ↑ Steffens, Heinz; Wegner, Waja; Willig, Katrin I. (2020-03-01). "In vivo STED microscopy: A roadmap to nanoscale imaging in the living mouse". Methods. 174: 42–48. doi: 10.1016/j.ymeth.2019.05.020 . ISSN   1095-9130. PMID   31132408.
  37. ↑ Steffens, Heinz; Mott, Alexander C.; Li, Siyuan; Wegner, Waja; Švehla, Pavel; Kan, Vanessa W. Y.; Wolf, Fred; Liebscher, Sabine; Willig, Katrin I. (2021). "Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity". Science Advances. 7 (24). Bibcode:2021SciA....7.2806S. doi:10.1126/sciadv.abf2806. ISSN   2375-2548. PMC   8189587 . PMID   34108204.
  38. ↑ Wegner, Waja; Steffens, Heinz; Gregor, Carola; Wolf, Fred; Willig, Katrin I. (2021-09-15). "Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture revealed by STED nanoscopy". bioRxiv: 2020.10.23.352195. doi: 10.1101/2020.10.23.352195 . S2CID   237538532.
  39. ↑ Westphal, V.; M. A. Lauterbach; A. Di Nicola; S. W. Hell (2007). "Dynamic far-field fluorescence nanoscopy". New J. Phys. 9 (12): 435. Bibcode:2007NJPh....9..435W. doi: 10.1088/1367-2630/9/12/435 .
  40. ↑ Lauterbach, M.A.; Chaitanya K. Ullal; Volker Westphal; Stefan W. Hell (2010). "Dynamic Imaging of Colloidal-Crystal Nanostructures at 200 Frames per Second". Langmuir. 26 (18): 14400–14404. doi: 10.1021/la102474p . PMID   20715873.
  41. ↑ Hotta, J. I.; E. Fron; P. Dedecker; K. P. F. Janssen; C. Li; K. Mullen; B. Harke; J. Buckers; S. W. Hell; J. Hofkens (2010). "Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores". J. Am. Chem. Soc. 132 (14): 5021–5023. doi:10.1021/ja100079w. hdl: 11858/00-001M-0000-0010-9310-1 . PMID   20307062.
  42. ↑ Vogelsang, J.; R. Kasper; C. Sreinhauer; B. Person; M. Heilemann; M. Sauer; P. Tinnedeld (2008). "Ein System aus Reduktions‐ und Oxidationsmittel verringert Photobleichen und Blinken von Fluoreszenzfarbstoffen". Angew. Chem. 120 (29): 5545–5550. doi:10.1002/ange.200801518.
  43. ↑ Vogelsang, J.; R. Kasper; C. Sreinhauer; B. Person; M. Heilemann; M. Sauer; P. Tinnedeld (2008). "A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes". Angew. Chem. Int. Ed. 47 (29): 5465–5469. doi:10.1002/anie.200801518. PMID   18601270.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.