Photoactivated localization microscopy

Last updated

Photo-activated localization microscopy (PALM or FPALM) [1] [2] and stochastic optical reconstruction microscopy (STORM) [3] are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) 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. [4] 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 However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes.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 (called activator), when excited near its absorption maximum, serves to reactivate the other molecule (called reporter) to the fluorescent state.

Contents

A growing number of dyes are used for PALM, STORM and related techniques, both organic fluorophores and fluorescent proteins. Some are compatible with live cell imaging, others allow faster acquisition or denser labeling. The choice of a particular fluorophore ultimately depends on the application and on its underlying photophysical properties. [5]

Both techniques have undergone significant technical developments, [6] in particular allowing multicolor imaging and the extension to three dimensions, with the best current axial resolution of 10 nm in the third dimension obtained using an interferometric approach with two opposing objectives collecting the fluorescence from the sample. [7]

Principle

Schematic representation of the reconstruction of a high resolution image by localizing the centroid of many low-resolution images of point-like light emitters

Conventional fluorescence microscopy is performed by selectively staining the sample with fluorescent molecules, either linked to antibodies as in immunohistochemistry or using fluorescent proteins genetically fused to the genes of interest. Typically, the more concentrated the fluorophores, the better the contrast of the fluorescence image.

A single fluorophore can be visualized under a microscope (or even under the naked eye [8] ) if the number of photons emitted is sufficiently high, and in contrast the background is low enough. The two dimensional image of a point source observed under a microscope is an extended spot, corresponding to the Airy disk (a section of the point spread function) of the imaging system. The ability to identify as two individual entities two closely spaced fluorophores is limited by the diffraction of light. This is quantified by Abbe’s criterion, stating that the minimal distance that allows resolving two point sources is given by

where is the wavelength of the fluorescent emission and NA is the numerical aperture of the microscope. The theoretical resolution limit at the shortest practical excitation wavelength is around 150 nm in the lateral dimension and approaching 400 nm in the axial dimension (if using an objective having a numerical aperture of 1.40 and the excitation wavelength is 400 nm).

However, if the emission from the two neighboring fluorescent molecules is made distinguishable, i.e. the photons coming from each of the two can be identified, then it is possible to overcome the diffraction limit. [9] Once a set of photons from a specific molecule is collected, it forms a diffraction-limited spot in the image plane of the microscope. The center of this spot can be found by fitting the observed emission profile to a known geometrical function, typically a Gaussian function in two dimensions. The error that is made in localizing the center of a point emitter scales to a first approximation as the inverse square root of the number of emitted photons, and if enough photons are collected it is easy to obtain a localization error much smaller than the original point spread function.

Multiple closely spaced emitters are indistinguishable. The position of a point source can be recovered only if the photons it emitted have been identified from those arising from the neighboring molecules Concept IdentificationAndLocalization pointemitters.png
Multiple closely spaced emitters are indistinguishable. The position of a point source can be recovered only if the photons it emitted have been identified from those arising from the neighboring molecules

The two steps of identification and localization of individual fluorescent molecules in a dense environment where many are present are at the basis of PALM, STORM and their development.

Although many approaches to molecular identification exist, the light-induced photochromism of selected fluorophores developed as the most promising approach to distinguish neighboring molecules by separating their fluorescent emission in time. By turning on stochastically sparse subsets of fluorophores with light of a specific wavelength, individual molecules can then be excited and imaged according to their spectra. To avoid the accumulation of active fluorophores in the sample, which would eventually degrade back to a diffraction-limited image, the spontaneously occurring phenomenon of photobleaching is exploited in PALM, whereas reversible switching between a fluorescent on-state and a dark off-state of a dye is exploited in STORM.

Photoactivation, localization and bleaching Schematics ActivationLocalizationBleaching PhotoActivatedLocalizationMicroscopy.tif
Photoactivation, localization and bleaching

In summary, PALM and STORM are based on collecting under a fluorescent microscope a large number of images each containing just a few active isolated fluorophores. The imaging sequence allows for the many emission cycles necessary to stochastically activate each fluorophore from a non-emissive (or less emissive) state to a bright state, and back to a non-emissive or bleached state. During each cycle, the density of activated molecules is kept low enough that the molecular images of individual fluorophores do not typically overlap.

Single CCD frame displaying individual fluorescent proteins excited in Total Internal Reflection FrameWithSingleMolecules.png
Single CCD frame displaying individual fluorescent proteins excited in Total Internal Reflection
Sequence of frames containing single molecule images Framesovertime.png
Sequence of frames containing single molecule images

Localization of individual fluorophores

In each image of the sequence, the position of a fluorophore is calculated with a precision typically greater than the diffraction limit - in the typical range of a few to tens of nm - and the resulting information of the position of the centers of all the localized molecules is used to build up the super-resolution PALM or STORM image.

The localization precision can be calculated according to the formula:

where N is the number of collected photons, a is the pixel size of the imaging detector, is the average background signal and is the standard deviation of the point spread function. [10] The requirement of localizing at the same time multiple fluorophores simultaneously over an extended area determines the reason why these methods are wide-field, employing as a detector a CCD, EMCCD or a CMOS camera.

The requirement for an enhanced signal-to-noise ratio to maximize localization precision determines the frequent combination of this concept with widefield fluorescent microscopes allowing optical sectioning, such as total internal reflection fluorescence microscopes (TIRF) and light sheet fluorescence microscopes.

Super-resolution image

The resolution of the final image is limited by the precision of each localization and the number of localizations, instead of by diffraction. The super resolution image is therefore a pointillistic representation of the coordinates of all the localized molecules. The super resolution image is commonly rendered by representing each molecule in the image plane as a two dimensional Gaussian with amplitude proportional to the number of photons collected, and the standard deviation depending on the localization precision.

Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. Scale bar in (A-E) indicates 1 mm. Scale bar in (F-H) indicates 50 nm. Journal.pbio.1000137.g002.png
Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. Scale bar in (A–E) indicates 1 μm. Scale bar in (F–H) indicates 50 nm.

Applications

Multicolor PALM/STORM

Strategies for multicolor localization microscopy. Left: spectral separation. Center: multiple activators (STORM). Right: Ratiometric imaging SuperResolutionMulticolorStrategies.png
Strategies for multicolor localization microscopy. Left: spectral separation. Center: multiple activators (STORM). Right: Ratiometric imaging

The peculiar photophysical properties of the fluorophores employed in PALM/STORM super resolution imaging pose both constraints and opportunities for multicolor imaging. Three strategies have emerged so far: excitation of spectrally separated fluorophores using an emission beamsplitter, [12] using of multiple activators/reporters in STORM mode [13] [14] and ratiometric imaging of spectrally close fluorophores. [15]

3D in PALM and STORM

Although originally developed as 2D (x,y) imaging methods, PALM and STORM have quickly developed into 3D (x,y,z) capable techniques. To determine the axial position of a single fluorophore in the sample the following approaches are currently being used: modification of the point spread function to introduce z-dependent features in the 2D (x,y) image (the most common approach is to introduce astigmatism in the PSF); multiplane detection, where the axial position is determined by comparing two images of the same PSF defocused one with respect to the other; interferometric determination of the axial position of the emitter using two opposed objectives and multiple detectors; [7] use of temporal focusing to confine the excitation/activation; use of light sheet excitation/activation to confine to a few hundred nanometers thick layer arbitrarily positioned along the z-plane within the sample.

Live cell imaging

The requirement for multiple cycles of activation, excitation and de-activation/bleaching would typically imply extended periods of time to form a PALM/STORM image, and therefore operation on a fixed sample. A number of works have been published as early as 2007 [16] performing PALM/STORM on live cells. The ability to perform live super-resolution imaging using these techniques ultimately depends on the technical limitations of collecting enough photons from a single emitter in a very short time. This depends both on the photophysical limitations of the probe as well as on the sensitivity of the detector employed. Relatively slow (seconds to tens of seconds) processes such as modification in the organization of focal adhesions have been investigated by means of PALM, [17] whereas STORM has allowed imaging of faster processes such as membrane diffusion of clathrin coated pits or mitochondrial fission/fusion processes. A promising application of live cell PALM is the use of photoactivation to perform high-density single-particle tracking (sptPALM [18] ), overcoming the traditional limitation of single particle tracking to work with systems displaying a very low concentration of fluorophores.

Nanophotonic interactions

While traditional PALM and STORM measurements are used to determine the physical structure of a sample, with the intensities of fluorescent events determining the certainty of the localization, these intensities can also be used to map fluorophore interactions with nanophotonic structures. This has been performed on both metallic (plasmonic) structures, such as gold nanorods, [19] [20] as well as semiconducting structures, such as silicon nanowires. [21] These approaches can either be used for fluorophores functionalized on the surface of the sample of interest (as for the plasmonic particle studies mentioned here), or randomly adsorbed onto the substrate surrounding the sample, allowing full 2D mapping of fluorophore-nanostructure interactions at all positions relative to the structure. [21]

These studies have found that, in addition to the standard uncertainty of localization due to the point spread function fitting, self-interference with light scattered by nanoparticles can lead to distortions or displacements of the imaged point spread functions, [20] [21] complicating the analysis of such measurements. These may be possible to limit, however, for example by incorporating metasurface masks which control the angular distribution of light permitted into the measurement system. [22]

Differences from STORM

PALM and STORM share a common fundamental principle, and numerous developments have tended to make the two techniques even more intertwined. Still, they differ in several technical details and a fundamental point. On the technical side, PALM is performed on a biological specimen using fluorophores expressed exogenously in the form of genetic fusion constructs to a photoactivatable fluorescent protein. STORM instead uses immunolabeling of endogenous molecules in the sample with antibodies tagged with organic fluorophores. In both cases the fluorophores are driven between an active-ON and an inactive-OFF state by light. In PALM, however, photoactivation and photobleaching confine the life of the fluorophore to a limited interval of time, and a continuous emission of the fluorophore is desirable in between without any fluorescence intermittency. In STORM stochastic photoblinking of the organic fluorophores (typically brighter than fluorescent proteins) was originally exploited to separate neighboring dyes. In this respect, the more robust the blinking, the higher the probability of distinguishing two neighbouring fluorophores.

In this respect, several research works have explored the potential of PALM to perform a quantitation of the number of fluorophores (and therefore proteins of interest) present in a sample by counting the activated fluorophores. [11] [23] [24] The approach used to treat the fluorescent dynamics of the fluorescent label used in the experiments will determine the final appearance of the super-resolution image, and the possibility of determining an unambiguous correspondence between a localization event and a protein in the sample.

Multimedia

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">STED microscopy</span> Technique in fluorescence 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. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

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">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).

<span class="mw-page-title-main">Fluorescence in the life sciences</span> Scientific investigative technique

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

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.

<span class="mw-page-title-main">Multifocal plane microscopy</span>

Multifocal plane microscopy (MUM), also known as multiplane microscopy or multifocus microscopy, is a form of light microscopy that allows the tracking of the 3D dynamics in live cells at high temporal and spatial resolution by simultaneously imaging different focal planes within the specimen. In this methodology, the light collected from the sample by an infinity-corrected objective lens is split into two paths. In each path the split light is focused onto a detector which is placed at a specific calibrated distance from the tube lens. In this way, each detector images a distinct plane within the sample. The first developed MUM setup was capable of imaging two distinct planes within the sample. However, the setup can be modified to image more than two planes by further splitting the light in each light path and focusing it onto detectors placed at specific calibrated distances. It has later been improved for imaging up to four distinct planes. To image a greater number of focal planes, simpler techniques based on image splitting optics have been developed. One example is by using a customized image splitting prism, which is capable of capturing up to 8 focal planes using only two cameras. Better yet, standard off-the-shelf partial beamsplitters can be used to construct a so-called z-splitter prism that allows simultaneous imaging of 9 individual focal planes using a single camera. Another technique called multifocus microscopy (MFM) uses diffractive Fourier optics to image up to 25 focal planes.

<span class="mw-page-title-main">Eric Betzig</span> American physicist

Robert Eric Betzig is an American physicist who works as a professor of physics and professor of molecular and cell biology at the University of California, Berkeley. He is also a senior fellow at the Janelia Farm Research Campus in Ashburn, Virginia.

Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can provide information about dynamic biological processes.

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.

<span class="mw-page-title-main">Fluorescence imaging</span> Type of non-invasive imaging technique

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.

<span class="mw-page-title-main">Suliana Manley</span> American biophysicist

Suliana Manley is an American biophysicist. Her research focuses on the development of high-resolution optical instruments, and their application in studying the organization and dynamics of proteins. She is a professor at École Polytechnique Fédérale de Lausanne and heads the Laboratory of Experimental Biophysics.

Ultrasound-switchable fluorescence (USF) imaging is a deep optics imaging technique. In last few decades, fluorescence microscopy has been highly developed to image biological samples and live tissues. However, due to light scattering, fluorescence microscopy is limited to shallow tissues. Since fluorescence is characterized by high contrast, high sensitivity, and low cost which is crucial to investigate deep tissue information, developing fluorescence imaging technique with high depth-to-resolution ratio would be promising.. Recently, ultrasound-switchable fluorescence imaging has been developed to achieve high signal-to-noise ratio (SNR) and high spatial resolution imaging without sacrificing image depth.

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. ↑ E. Betzig; G. H. Patterson; R. Sougrat; O. W. Lindwasser; S. Olenych; J. S. Bonifacino; M. W. Davidson; J. Lippincott-Schwartz; H. F. Hess (2006). "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution". Science. 313 (5793): 1642–1645. Bibcode:2006Sci...313.1642B. doi: 10.1126/science.1127344 . PMID   16902090.
  2. ↑ S. T. Hess; T. P. Giriajan; M. D. Mason (2006). "Ultra-high resolution imaging by Fluorescence Photoactivation Localization Microscopy". Biophysical Journal. 91 (11): 4258–4272. Bibcode:2006BpJ....91.4258H. doi:10.1529/biophysj.106.091116. PMC   1635685 . PMID   16980368.
  3. ↑ M. J. Rust; M. Bates; X. Zhuang (2006). "Sub diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)". Nature Methods. 3 (20): 793–796. doi:10.1038/nmeth929. PMC   2700296 . PMID   16896339.
  4. ↑ "Method of the Year 2008". Nature Methods. 6 (1): 1–109. 2009. doi: 10.1038/nmeth.f.244 .
  5. ↑ Ha, Taekjip & Tinnefeld, Philip (2012). "Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging". Annual Review of Physical Chemistry. 63 (1): 595–617. Bibcode:2012ARPC...63..595H. doi:10.1146/annurev-physchem-032210-103340. PMC   3736144 . PMID   22404588.
  6. ↑ Bo Huang and Hazen Babcock and Xiaowei Zhuang (2010). "Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells". Cell. 143 (7): 1047–58. doi:10.1016/j.cell.2010.12.002. PMC   3272504 . PMID   21168201.
  7. 1 2 Shtengel, Gleb and Galbraith, James A. and Galbraith, Catherine G. and Lippincott-Schwartz, Jennifer and Gillette, Jennifer M. and Manley, Suliana and Sougrat, Rachid and Waterman, Clare M. and Kanchanawong, Pakorn and Davidson, Michael W. and Fetter, Richard D. and Hess, Harald F. (2009). "Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure". Proceedings of the National Academy of Sciences. 106 (9): 3125–3130. Bibcode:2009PNAS..106.3125S. doi: 10.1073/pnas.0813131106 . PMC   2637278 . PMID   19202073.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ↑ W. E. Moerner; D. P. Fromm (2003). "Methods of single-molecule fluorescence spectroscopy and individual fluorescence probes". Review of Scientific Instruments. 74 (8): 3597–3619. Bibcode:2003RScI...74.3597M. doi:10.1063/1.1589587. S2CID   11615310.
  9. ↑ E. Betzig (1995). "Proposed Method for molecular optical imaging". Optics Letters. 20 (3): 237–239. Bibcode:1995OptL...20..237B. doi:10.1364/OL.20.000237. PMID   19859146.
  10. ↑ K. I. Mortensen; L S. Churchman; J. A. Spudich; H. Flyvbjerg (2010). "Optimized localization analysis for single-molecule tracking and super-resolution microscopy". Nature Methods. 7 (5): 377–381. doi:10.1038/nmeth.1447. PMC   3127582 . PMID   20364147.
  11. 1 2 Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS, et al. (2009). "Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy". PLOS Biology. 7 (6): e1000137. doi: 10.1371/journal.pbio.1000137 . PMC   2691949 . PMID   19547746.
  12. ↑ Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, Olenych S, Davidson MW, Betzig E (2007). "Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes". Proceedings of the National Academy of Sciences. 104 (51): 20308–20313. Bibcode:2007PNAS..10420308S. doi: 10.1073/pnas.0710517105 . PMC   2154427 . PMID   18077327.
  13. ↑ M Bates; B Huang; GT Dempsey; X Zhuang (2007). "Multicolor Super Resolution Imaging with Photo-Switchable fluorescent probes". Science. 317 (5845): 1749–1753. Bibcode:2007Sci...317.1749B. doi:10.1126/science.1146598. PMC   2633025 . PMID   17702910.
  14. ↑ Bock, H.; et al. (2007). "Two-color far-field fluorescence nanoscopy based on photoswitchable emitters". Applied Physics B. 88 (2): 161–165. Bibcode:2007ApPhB..88..161B. doi:10.1007/s00340-007-2729-0. S2CID   122146697.
  15. ↑ Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C, Folling J, Jakobs S, Schonle A, Hell SW, Eggeling C (2010). "Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength". Biophysical Journal. 99 (8): 2686–2694. Bibcode:2010BpJ....99.2686T. doi:10.1016/j.bpj.2010.08.012. PMC   2956215 . PMID   20959110.
  16. ↑ Hess, Samuel T. and Gould, Travis J. and Gudheti, Manasa V. and Maas, Sarah A. and Mills, Kevin D. and Zimmerberg, Joshua (2007). "Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories". Proceedings of the National Academy of Sciences. 104 (44): 17370–17375. Bibcode:2007PNAS..10417370H. doi: 10.1073/pnas.0708066104 . PMC   2077263 . PMID   17959773.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ↑ Shroff, H., C. G. Galbraith, J. A. Galbraith, and E. Betzig (2008). "Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics". Nature Methods. 5 (44): 417–423. doi:10.1038/nmeth.1202. PMC   5225950 . PMID   18408726.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  18. ↑ S Manley; J M Gillette; G H Patterson; H Shroff; H F Hess; E Betzig; J Lippincott-Schwartz (2008). "High-density mapping of single-molecule trajectories with photoactivated localization microscopy". Nature Methods. 5 (2): 155–157. doi:10.1038/nmeth.1176. PMID   18193054. S2CID   1101468.
  19. ↑ Zhou, Xiaochun; Andoy, Nesha May; Liu, Guokun; Choudhary, Eric; Han, Kyu-Sung; Shen, Hao; Chen, Peng (2012). "Quantitative super-resolution imaging uncovers reactivity patterns on single nanocatalysts". Nature Nanotechnology. 7 (4): 237–241. Bibcode:2012NatNa...7..237Z. doi:10.1038/nnano.2012.18. ISSN   1748-3387. PMID   22343380. S2CID   4654704.
  20. 1 2 Lin, Hongzhen; Centeno, Silvia P.; Su, Liang; Kenens, Bart; Rocha, Susana; Sliwa, Michel; Hofkens, Johan; Uji-i, Hiroshi (2012). "Mapping of Surface-Enhanced Fluorescence on Metal Nanoparticles using Super-Resolution Photoactivation Localization Microscopy". ChemPhysChem. 13 (4): 973–981. doi:10.1002/cphc.201100743. ISSN   1439-4235. PMID   22183928.
  21. 1 2 3 Johlin, Eric; Solari, Jacopo; Mann, Sander A.; Wang, Jia; Shimizu, Thomas S.; Garnett, Erik C. (2016). "Super-resolution imaging of light–matter interactions near single semiconductor nanowires". Nature Communications. 7: 13950. Bibcode:2016NatCo...713950J. doi:10.1038/ncomms13950. ISSN   2041-1723. PMC   5187462 . PMID   27996010.
  22. ↑ Backlund, Mikael P.; Arbabi, Amir; Petrov, Petar N.; Arbabi, Ehsan; Saurabh, Saumya; Faraon, Andrei; Moerner, W. E. (2016). "Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask" (PDF). Nature Photonics. 10 (7): 459–462. Bibcode:2016NaPho..10..459B. doi:10.1038/nphoton.2016.93. ISSN   1749-4885. PMC   5001689 . PMID   27574529.
  23. ↑ P Annibale; S Vanni; M Scarselli; U Rothlisberger; A Radenovic (2011). "Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking". PLOS ONE. 6 (7): p.e22678, 07. Bibcode:2011PLoSO...622678A. doi: 10.1371/journal.pone.0022678 . PMC   3144238 . PMID   21818365.
  24. ↑ Lee, Sang-Hyuk and Shin, Jae Yen and Lee, Antony and Bustamante, Carlos (2012). "Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM)". Proceedings of the National Academy of Sciences. 109 (43): 17436–17441. Bibcode:2012PNAS..10917436L. doi: 10.1073/pnas.1215175109 . PMC   3491528 . PMID   23045631.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  25. ↑ Izeddin I, Specht CG, Lelek M, Darzacq X, Triller A, et al. (2011). "Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe". PLOS ONE. 6 (1): e15611. Bibcode:2011PLoSO...615611I. doi: 10.1371/journal.pone.0015611 . PMC   3022016 . PMID   21264214.
  26. ↑ Tatavarty V, Kim E, Rodionov V, Yu J (2009). "Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging". PLOS ONE. 4 (11): e7724. Bibcode:2009PLoSO...4.7724T. doi: 10.1371/journal.pone.0007724 . PMC   2771285 . PMID   19898630.
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.