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Super-resolution microscopy is a series of techniques in optical
microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
that allow such images to have resolutions higher than those imposed by the
diffraction limit The resolution of an optical imaging system a microscope, telescope, or camera can be limited by factors such as imperfections in the lenses or misalignment. However, there is a principal limit to the resolution of any optical system, due to t ...
, which is due to the
diffraction Diffraction is defined as the interference or bending of waves around the corners of an obstacle or through an aperture into the region of geometrical shadow of the obstacle/aperture. The diffracting object or aperture effectively becomes a s ...
of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that utilize the Pendry Superlens and
near field scanning optical microscopy Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field Diffraction-limited system, resolution limit by exploiting the prop ...
) or on the
far-field The near field and far field are regions of the electromagnetic (EM) field around an object, such as a transmitting antenna, or the result of radiation scattering off an object. Non-radiative ''near-field'' behaviors dominate close to the ante ...
. Among techniques that rely on the latter are those that improve the resolution only modestly (up to about a factor of two) beyond the diffraction-limit, such as
confocal microscopy Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a ...
with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment (e.g. re-scan microscopy, pixel reassignment), the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI. There are two major groups of methods for super-resolution microscopy in the far-field that can improve the resolution by a much larger factor: # Deterministic super-resolution: the most commonly used emitters in biological microscopy,
fluorophores A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...
, show a nonlinear response to excitation, which can be exploited to enhance resolution. Such methods include STED,
GSD GSD may refer to: Schools * Georgia School for the Deaf, in Cave Spring, Georgia, United States * Harvard Graduate School of Design Science and medicine * Genetic significant dose * Global Species Database * Glutathione synthetase deficiency * ...
,
RESOLFT RESOLFT, an acronym for REversible Saturable OpticaL Fluorescence 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 diffrac ...
and SSIM. # Stochastic super-resolution: the chemical complexity of many molecular light sources gives them a complex temporal behavior, which can be used to make several nearby fluorophores emit light at separate times and thereby become resolvable in time. These methods include
Super-resolution optical fluctuation imaging Super-resolution optical fluctuation imaging (SOFI) is a post-processing method for the calculation of super-resolved images from recorded image time series that is based on the temporal correlations of independently fluctuating fluorescent emitters ...
(SOFI) and all single-molecule localization methods (SMLM), such as SPDM, SPDMphymod,
PALM Palm most commonly refers to: * Palm of the hand, the central region of the front of the hand * Palm plants, of family Arecaceae **List of Arecaceae genera * Several other plants known as "palm" Palm or Palms may also refer to: Music * Palm (ba ...
, FPALM, STORM, and dSTORM. On 8 October 2014, the
Nobel Prize in Chemistry ) , image = Nobel Prize.png , alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
was awarded to Eric Betzig,
W.E. Moerner William Esco Moerner (born June 24, 1953) is an American physical chemist and chemical physicist with current work in the biophysics and imaging of single molecules. He is credited with achieving the first optical detection and spectroscopy of ...
and
Stefan Hell Stefan Walter Hell HonFRMS (: born 23 December 1962) is a Romanian-German physicist and one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany. He received the Nobel Prize in Chemistry in 2014 "for the d ...
for "the development of super-resolved
fluorescence microscopy 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 microscop ...
", which brings "
optical microscopy Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultravio ...
into the nanodimension". The different modalities of super-resolution microscopy are increasingly being adopted by the biomedical research community, and these techniques are becoming indispensable tools to understanding biological function at the molecular level.


History

By 1978, the first theoretical ideas had been developed to break the Abbe limit, which called for using a 4Pi microscope as a confocal laser-scanning fluorescence microscope where the light is focused from all sides to a common focus that is used to scan the object by 'point-by-point' excitation combined with 'point-by-point' detection. However the publication from 1978 had drawn an improper physical conclusion (i.e. a point-like spot of light) and had completely missed the axial resolution increase as the actual benefit of adding the other side of the solid angle. Some of the following information was gathered (with permission) from a chemistry blog's review of sub-diffraction microscopy techniques. In 1986, a super-resolution optical microscope based on stimulated emission was patented by Okhonin.V.A. Okhonin, Method of investigating specimen microstructure
Patent SU 1374922
priority date 10 April 1986
Published on July 30, 1991
Soviet Patents Abstracts, Section EI, Week 9218, Derwent Publications Ltd., London, GB; Class S03, p. 4. Cited by patent
US 5394268 A
(1993) an
US RE38307 E1
(1995). From th
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...".


Super-resolution techniques


Photon tunneling microscopy (PTM)


Local enhancement / ANSOM / optical nano-antennas


Near-field optical random mapping (NORM) microscopy

Near-field optical random mapping (NORM) microscopy is a method of optical near-field acquisition by a far-field microscope through the observation of nanoparticles' Brownian motion in an immersion liquid. NORM utilizes object surface scanning by stochastically moving nanoparticles. Through the microscope, nanoparticles look like symmetric round spots. The spot width is equivalent to the point spread function (~ 250 nm) and is defined by the microscope resolution. Lateral coordinates of the given particle can be evaluated with a precision much higher than the resolution of the microscope. By collecting the information from many frames one can map out the near field intensity distribution across the whole field of view of the microscope. In comparison with NSOM and ANSOM this method does not require any special equipment for tip positioning and has a large field of view and a depth of focus. Due to the large number of scanning "sensors" one can achieve image acquisition in a shorter time.


4Pi

A 4Pi microscope is a laser-scanning
fluorescence microscope 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 microsco ...
with an improved
axial Axial may refer to: * one of the anatomical directions describing relationships in an animal body * In geometry: :* a geometric term of location :* an axis of rotation * In chemistry, referring to an axial bond * a type of modal frame, in music * ...
resolution. The typical value 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 Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a ...
. The improvement in resolution is achieved by using two opposing objective lenses, both of which are focused to the same geometric location. Also, the difference in
optical path length In optics, optical path length (OPL, denoted ''Λ'' in equations), also known as optical length or optical distance, is the product of the geometric length of the optical path followed by light and the refractive index of homogeneous medium throu ...
through each of the two objective lenses is carefully minimized. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides, and the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The
solid angle In geometry, a solid angle (symbol: ) is a measure of the amount of the field of view from some particular point that a given object covers. That is, it is a measure of how large the object appears to an observer looking from that point. The poi ...
\Omega that is used for illumination and detection is increased and approaches the ideal case, where the sample is illuminated and detected from all sides simultaneously. Up to now, the best quality in a 4Pi microscope has been reached in conjunction with
STED 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 ...
in fixed cells and
RESOLFT RESOLFT, an acronym for REversible Saturable OpticaL Fluorescence 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 diffrac ...
microscopy with switchable proteins in living cells.


Structured illumination microscopy (SIM)

Structured illumination microscopy (SIM) enhances spatial resolution by collecting information from frequency space outside the observable region. This process is done in reciprocal space: the
Fourier transform A Fourier transform (FT) is a mathematical transform that decomposes functions into frequency components, which are represented by the output of the transform as a function of frequency. Most commonly functions of time or space are transformed ...
(FT) of an SI image contains superimposed additional information from different areas of reciprocal space; with several frames where the illumination is shifted by some phase, it is possible to computationally separate and reconstruct the FT image, which has much more resolution information. The reverse FT returns the reconstructed image to a super-resolution image. SIM microscopy could potentially replace
electron microscopy An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
as a tool for some medical diagnoses. These include diagnosis of kidney disorders, kidney cancer, and blood diseases. Although the term "structured illumination microscopy" was coined by others in later years, Guerra (1995) first published results in which light patterned by a 50 nm pitch grating illuminated a second grating of pitch 50 nm, with the gratings rotated with respect to each other by the angular amount needed to achieve magnification. Although the illuminating wavelength was 650 nm, the 50 nm grating was easily resolved. This showed a nearly 5-fold improvement over the Abbe resolution limit of 232 nm that should have been the smallest obtained for the numerical aperture and wavelength used. In further development of this work, Guerra showed that super-resolved lateral topography is attained by phase-shifting the evanescent field. Several U.S. patentsU.S. Patent Number 5,774,221, Apparatus and methods for providing phase-controlled evanescent illumination (1996)Number 5,666,197, Apparatus and methods employing phase control and analysis of evanescent for imaging and metrology of subwavelength lateral surface topography (1996)
an
Number 5,715,059, Dark field, photon tunneling systems and methods (1996)
/ref> were issued to Guerra individually, or with colleagues, and assigned to the
Polaroid Corporation Polaroid is an American company best known for its instant film and cameras. The company was founded in 1937 by Edwin H. Land, to exploit the use of its Polaroid polarizing polymer. Land ran the company until 1981. Its peak employment was 21,0 ...
. Licenses to this technology were procured by Dyer Energy Systems, Calimetrics Inc., and Nanoptek Corp. for use of this super-resolution technique in optical data storage and microscopy. cell nuclei The cell nucleus (pl. nuclei; from Latin or , meaning ''kernel'' or ''seed'') is a membrane-bound organelle found in eukaryotic cells. Eukaryotic cells usually have a single nucleus, but a few cell types, such as mammalian red blood cells, ha ...
and
mitotic In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is maintai ...
stages recorded with 3D-SIM."> File:3D-SIM-1 NPC Confocal vs 3D-SIM.jpg, Comparison confocal microscopy – 3D-SIM File:3D-SIM-2 Nucleus prophase 3d rotated.jpg, Cell nucleus in
prophase Prophase () is the first stage of cell division in both mitosis and meiosis. Beginning after interphase, DNA has already been replicated when the cell enters prophase. The main occurrences in prophase are the condensation of the chromatin ret ...
from various angles File:3D-SIM-3 Prophase 3 color.jpg, Two mouse cell nuclei in prophase. File:3D-SIM-4 Anaphase 3 color.jpg, mouse cell in
telophase Telophase () is the final stage in both meiosis and mitosis in a eukaryotic cell. During telophase, the effects of prophase and prometaphase (the nucleolus and nuclear membrane disintegrating) are reversed. As chromosomes reach the cell poles, ...


Spatially modulated illumination (SMI)

One implementation of structured illumination is known as spatially modulated illumination (SMI). Like standard structured illumination, the SMI technique modifies the point spread function (PSF) of a microscope in a suitable manner. In this case however, "the optical resolution itself is not enhanced"; instead structured illumination is used to maximize the precision of
distance Distance is a numerical or occasionally qualitative measurement of how far apart objects or points are. In physics or everyday usage, distance may refer to a physical length or an estimation based on other criteria (e.g. "two counties over"). ...
measurements of fluorescent objects, to "enable size measurements at molecular dimensions of a few tens of nanometers". The Vertico SMI microscope achieves structured illumination by using one or two opposing interfering laser beams along the axis. The object being imaged is then moved in high-precision steps through the wave field, or the wave field itself is moved relative to the object by phase shifts. This results in an improved axial size and distance resolution. SMI can be combined with other super resolution technologies, for instance with 3D LIMON or LSI- TIRF as a
total internal reflection Total internal reflection (TIR) is the optical phenomenon in which waves arriving at the interface (boundary) from one medium to another (e.g., from water to air) are not refracted into the second ("external") medium, but completely reflect ...
interferometer with laterally structured illumination (this last instrument and technique is essentially a phase-shifted photon tunneling microscope, which employs a total internal reflection light microscope with phase-shifted evanescent field (Guerra, 1996)). This SMI technique allows one to acquire light-optical images of autofluorophore distributions in sections from human eye tissue with previously unmatched optical resolution. Use of three different excitation wavelengths (488, 568, and 647 nm), enables one to gather spectral information about the autofluorescence signal. This has been used to examine human eye tissue affected by
macular degeneration Macular degeneration, also known as age-related macular degeneration (AMD or ARMD), is a medical condition which may result in blurred or no vision in the center of the visual field. Early on there are often no symptoms. Over time, however, so ...
.


Deterministic functional techniques

REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy is an
optical microscopy Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultravio ...
with very high resolution that can image details in samples that cannot be imaged with conventional or
confocal microscopy Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a ...
. Within RESOLFT the principles of
STED 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 ...
and
GSD microscopy Ground state depletion microscopy (GSD 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 opt ...
are generalized. Also, there are techniques with other concepts than RESOLFT or SSIM. For example,
fluorescence microscopy 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 microscop ...
using the optical
AND gate The AND gate is a basic digital logic gate that implements logical conjunction (∧) from mathematical logic AND gate behaves according to the truth table. A HIGH output (1) results only if all the inputs to the AND gate are HIGH (1). If not al ...
property of
nitrogen-vacancy center The nitrogen-vacancy center (N-V center or NV center) is one of numerous point defects in diamond. Its most explored and useful property is its photoluminescence, which allows observers to read out its spin-state. The NV center's electron spin, loc ...
, or super-resolution by Stimulated Emission of Thermal Radiation (SETR), which uses the intrinsic super-linerarities of the
Black-Body radiation Black-body radiation is the thermal electromagnetic radiation within, or surrounding, a body in thermodynamic equilibrium with its environment, emitted by a black body (an idealized opaque, non-reflective body). It has a specific, continuous spe ...
and expands the concept of super-resolution beyond microscopy.


Stimulated emission depletion (STED)

Stimulated emission depletion 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 p ...
(STED) uses two laser pulses, the excitation pulse for excitation of the
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
s to their fluorescent state and the STED pulse for the de-excitation of fluorophores by means of
stimulated emission Stimulated emission is the process by which an incoming photon of a specific frequency can interact with an excited atomic electron (or other excited molecular state), causing it to drop to a lower energy level. The liberated energy transfers to th ...
. In practice, the excitation laser pulse is first applied whereupon a STED pulse soon follows (STED without pulses using continuous wave lasers is also used). Furthermore, the STED pulse is modified in such a way so that it features a zero-intensity spot that coincides with the excitation focal spot. Due to the non-linear dependence of the stimulated emission rate on the intensity of the STED beam, all the fluorophores around the focal excitation spot will be in their off state (the ground state of the fluorophores). By scanning this focal spot, one retrieves the image. The
full width at half maximum In a distribution, full width at half maximum (FWHM) is the difference between the two values of the independent variable at which the dependent variable is equal to half of its maximum value. In other words, it is the width of a spectrum curve mea ...
(FWHM) of the point spread function (PSF) of the excitation focal spot can theoretically be compressed to an arbitrary width by raising the intensity of the STED pulse, according to equation (). : \Delta r \approx \frac   () : where ∆r is the lateral resolution, ∆ is the FWHM of the diffraction limited PSF, ''I''max is the peak intensity of the STED laser, and I_ is the threshold intensity needed in order to achieve saturated emission depletion. The main disadvantage of STED, which has prevented its widespread use, is that the machinery is complicated. On the one hand, the image acquisition speed is relatively slow for large fields of view because of the need to scan the sample in order to retrieve an image. On the other hand, it can be very fast for smaller fields of view: recordings of up to 80 frames per second have been shown. Due to a large ''Is'' value associated with STED, there is the need for a high-intensity excitation pulse, which may cause damage to the sample.


Ground state depletion (GSD)

Ground state depletion microscopy (GSD microscopy) uses the
triplet state In quantum mechanics, a triplet is a quantum state of a system with a spin of quantum number =1, such that there are three allowed values of the spin component, = −1, 0, and +1. Spin, in the context of quantum mechanics, is not a mechanical r ...
of a fluorophore as the off-state and the singlet state as the on-state, whereby an excitation laser is used to drive the fluorophores at the periphery of the
singlet state In quantum mechanics, a singlet state usually refers to a system in which all electrons are paired. The term 'singlet' originally meant a linked set of particles whose net angular momentum is zero, that is, whose overall spin quantum number s=0. A ...
molecule to the triplet state. This is much like STED, where the off-state is the ground state of fluorophores, which is why equation () also applies in this case. The I_ value is smaller than in STED, making super-resolution imaging possible at a much smaller laser intensity. Compared to STED, though, the fluorophores used in GSD are generally less photostable; and the saturation of the triplet state may be harder to realize.


Saturated structured illumination microscopy (SSIM)

Saturated structured-illumination microscopy (SSIM) exploits the nonlinear dependence of the emission rate of fluorophores on the intensity of the excitation laser. By applying a sinusoidal illumination pattern with a peak intensity close to that needed in order to saturate the fluorophores in their fluorescent state, one retrieves Moiré fringes. The fringes contain high order spatial information that may be extracted by computational techniques. Once the information is extracted, a super-resolution image is retrieved. SSIM requires shifting the illumination pattern multiple times, effectively limiting the temporal resolution of the technique. In addition there is the need for very photostable fluorophores, due to the saturating conditions, which inflict radiation damage on the sample and restrict the possible applications for which SSIM may be used. Examples of this microscopy are shown under section Structured illumination microscopy (SIM): images of cell nuclei and mitotic stages recorded with 3D-SIM Microscopy.


Stochastic functional techniques


Localization microscopy

Single-molecule localization microscopy (SMLM) summarizes all microscopical techniques that achieve super-resolution by isolating emitters and fitting their images with the point spread function (PSF). Normally, the width of the point spread function (~ 250 nm) limits resolution. However, given an isolated emitter, one is able to determine its location with a precision only limited by its intensity according to equation (). : \Delta \mathrm \approx \frac   () : where Δloc is the localization precision, Δ is the FWHM (full width at half maximum) of the PSF and N is the number of collected photons. This fitting process can only be performed reliably for isolated emitters (see
Deconvolution In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a deco ...
), and interesting biological samples are so densely labeled with emitters that fitting is impossible when all emitters are active at the same time. SMLM techniques solve this dilemma by activating only a sparse subset of emitters at the same time, localizing these few emitters very precisely, deactivating them and activating another subset. Considering background and camera pixelation, and using Gaussian approximation for the point spread function (
Airy disk In optics, the Airy disk (or Airy disc) and Airy pattern are descriptions of the best- focused spot of light that a perfect lens with a circular aperture can make, limited by the diffraction of light. The Airy disk is of importance in physics, ...
) of a typical microscope, the theoretical resolution is proposed by Thompson et al. and fine-tuned by Mortensen et al.: : \sigma^2 = \frac(\frac+\frac) : where : * ''σ'' is the Gaussian
standard deviation In statistics, the standard deviation is a measure of the amount of variation or dispersion of a set of values. A low standard deviation indicates that the values tend to be close to the mean (also called the expected value) of the set, whil ...
of the center locations of the same molecule if measured multiple times (e.g. frames of a video). (unit m) : * ''σ''PSF is the Gaussian standard deviation of the point spread function, whose FWHM following the
Ernst Abbe Ernst Karl Abbe HonFRMS (23 January 1840 – 14 January 1905) was a German physicist, optical scientist, entrepreneur, and social reformer. Together with Otto Schott and Carl Zeiss, he developed numerous optical instruments. He was also a c ...
equation ''d = λ/(2 N.A.)''. (unit m) : * ''a'' is the size of each image pixel. (unit m) : * ''N''sig is the photon counts of the total PSF over all pixels of interest. (unitless) : * ''N''bg the average background photon counts per pixel (dark counts already removed), which is approximated to be the square of the Gaussian
standard deviation In statistics, the standard deviation is a measure of the amount of variation or dispersion of a set of values. A low standard deviation indicates that the values tend to be close to the mean (also called the expected value) of the set, whil ...
of the
Poisson distribution In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time or space if these events occur with a known co ...
background noise of each pixel over time or standard deviation of all pixels with background noise only, ''σ''bg2. The larger the ''σ''bg2, the better the approximation (e.g. good for ''σ''bg2 >10, excellent for ''σ''bg2 >1000). (unitless) : * Resolution FWHM is ~2.355 times the Gaussian
standard deviation In statistics, the standard deviation is a measure of the amount of variation or dispersion of a set of values. A low standard deviation indicates that the values tend to be close to the mean (also called the expected value) of the set, whil ...
. Generally, localization microscopy is performed with fluorophores. Suitable fluorophores (e.g. for STORM) reside in a non-fluorescent dark state for most of the time and are activated stochastically, typically with an excitation laser of low intensity. A readout laser stimulates fluorescence and bleaches or photoswitches the fluorophores back to a dark state, typically within 10–100 ms. In points accumulation for imaging in nanoscale topography (PAINT), the fluorophores are nonfluorescent before binding and fluorescent after. The photons emitted during the fluorescent phase are collected with a camera and the resulting image of the fluorophore (which is distorted by the PSF) can be fitted with very high precision, even on the order of a few Angstroms. Repeating the process several thousand times ensures that all fluorophores can go through the bright state and are recorded. A computer then reconstructs a super-resolved image. The desirable traits of fluorophores used for these methods, in order to maximize the resolution, are that they should be bright. That is, they should have a high extinction coefficient and a high
quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system. Applications Fluorescence spectroscopy The fluorescence quantum yield is defined as the ratio of the numb ...
. They should also possess a high contrast ratio (ratio between the number of photons emitted in the light state and the number of photons emitted in the dark state). Also, a densely labeled sample is desirable, according to the Nyquist criteria. The multitude of localization microscopy methods differ mostly in the type of fluorophores used.


Spectral precision distance microscopy (SPDM)

A single, tiny source of light can be located much better than the resolution of a microscope usually allows for: although the light will produce a blurry spot, computer algorithms can be used to accurately calculate the center of the blurry spot, taking into account the point spread function of the microscope, the noise properties of the detector, etc. However, this approach does not work when there are too many sources close to each other: the sources then all blur together. Spectral precision distance microscopy (SPDM) is a family of localizing techniques in
fluorescence microscopy 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 microscop ...
which gets around the problem of there being many sources by measuring just a few sources at a time, so that each source is "optically isolated" from the others (i.e., separated by more than the microscope's resolution, typically ~200-250 nm), if the particles under examination have different spectral signatures, so that it is possible to look at light from just a few molecules at a time by using the appropriate light sources and filters. This achieves an effective optical resolution several times better than the conventional optical resolution that is represented by the half-width of the main maximum of the effective point image function. The structural resolution achievable using SPDM can be expressed in terms of the smallest measurable distance between two punctiform particles of different spectral characteristics ("topological resolution"). Modeling has shown that under suitable conditions regarding the precision of localization, particle density, etc., the "topological resolution" corresponds to a "
space frequency Frequency-shift keying (FSK) is a frequency modulation scheme in which digital information is transmitted through discrete frequency changes of a carrier signal. The technology is used for communication systems such as telemetry, weather ball ...
" that, in terms of the classical definition, is equivalent to a much improved optical resolution. Molecules can also be distinguished in even more subtle ways based on fluorescent lifetime and other techniques. An important application is in genome research (study of the functional organization of the
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...
). Another important area of use is research into the structure of membranes.


= SPDMphymod

= Localization microscopy for many standard fluorescent dyes like GFP, Alexa dyes, and
fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
molecules is possible if certain photo-physical conditions are present. With this so-called physically modifiable fluorophores (SPDMphymod) technology, a single laser wavelength of suitable intensity is sufficient for nanoimaging in contrast to other localization microscopy technologies that need two laser wavelengths when special photo-switchable/photo-activatable fluorescence molecules are used. A further example of the use of SPDMphymod is an analysis of
Tobacco mosaic virus ''Tobacco mosaic virus'' (TMV) is a positive-sense single-stranded RNA virus species in the genus ''Tobamovirus'' that infects a wide range of plants, especially tobacco and other members of the family Solanaceae. The infection causes characteri ...
(TMV) particles or the study of virus–cell interaction. Based on singlet–triplet state transitions it is crucial for SPDMphymod that this process is ongoing and leading to the effect that a single molecule comes first into a very long-living reversible dark state (with half-life of as much as several seconds) from which it returns to a fluorescent state emitting many photons for several milliseconds before it returns into a very long-living, so-called irreversible dark state. SPDMphymod microscopy uses fluorescent molecules that emit the same spectral light frequency but with different spectral signatures based on the flashing characteristics. By combining two thousands images of the same cell, it is possible, using laser optical precision measurements, to record localization images with significantly improved optical resolution. Standard fluorescent dyes already successfully used with the SPDMphymod technology are GFP, RFP, YFP, Alexa 488, Alexa 568, Alexa 647, Cy2, Cy3, Atto 488 and
fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
.


Cryogenic optical localization in 3D (COLD)

Cryogenic Optical Localization in 3D (COLD) is a method that allows localizing multiple fluorescent sites within a single small- to medium-sized biomolecule with Angstrom-scale resolution. The localization precision in this approach is enhanced because the slower photochemistry at low temperatures leads to a higher number of photons that can be emitted from each fluorophore before photobleaching. As a result, cryogenic stochastic localization microscopy achieves the sub-molecular resolution required to resolve the 3D positions of several fluorophores attached to a small protein. By employing algorithms known from electron microscopy, the 2D projections of fluorophores are reconstructed into a 3D configuration. COLD brings fluorescence microscopy to its fundamental limit, depending on the size of the label. The method can also be combined with other structural biology techniques—such as X-ray crystallography, magnetic resonance spectroscopy, and electron microscopy—to provide valuable complementary information and specificity.


Binding-activated localization microscopy (BALM)

Binding-activated localization microscopy (BALM) is a general concept for single-molecule localization microscopy (SMLM): super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and a dye. By careful adjustment of the chemical environment—leading to local, reversible DNA melting and hybridization control over the fluorescence signal—DNA-binding dye molecules can be introduced. Intercalating and minor-groove binding DNA dyes can be used to register and optically isolate only a few DNA-binding dye signals at a time. DNA structure fluctuation-assisted BALM (fBALM) has been used to nanoscale differences in nuclear architecture, with an anticipated structural resolution of approximately 50 nm. Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations. Recently, the significant enhancement of fluorescence quantum yield of NIAD-4 upon binding to an
amyloid Amyloids are aggregates of proteins characterised by a fibrillar morphology of 7–13 nm in diameter, a beta sheet (β-sheet) secondary structure (known as cross-β) and ability to be stained by particular dyes, such as Congo red. In the huma ...
was exploited for BALM imaging of amyloid fibrils and oligomers.


STORM, PALM, and FPALM

Stochastic optical reconstruction microscopy (STORM), photo activated localization microscopy (PALM), and fluorescence photo-activation localization microscopy (FPALM) are super-resolution imaging techniques that utilize sequential activation and time-resolved localization of photoswitchable fluorophores to create high resolution images. During imaging, only an optically resolvable subset of fluorophores is activated to a fluorescent state at any given moment, such that the position of each fluorophore can be determined with high precision by finding the centroid positions of the single-molecule images of a particular fluorophore. One subset of fluorophores is subsequently deactivated, and another subset is activated and imaged. Iteration of this process allows numerous fluorophores to be localized and a super-resolution image to be constructed from the image data. These three methods were published independently over a short period of time, and their principles are identical. STORM was originally described using Cy5 and Cy3 dyes attached to nucleic acids or proteins, while PALM and FPALM were described using photoswitchable fluorescent proteins. In principle any photoswitchable fluorophore can be used, and STORM has been demonstrated with a variety of different probes and labeling strategies. Using stochastic photoswitching of single fluorophores, such as Cy5, STORM can be performed with a single red laser excitation source. The red laser both switches the Cy5 fluorophore to a dark state by formation of an adduct and subsequently returns the molecule to the fluorescent state. Many other dyes have been also used with STORM. In addition to single fluorophores, dye-pairs consisting of an activator fluorophore (such as Alexa 405, Cy2, or Cy3) and a photoswitchable reporter dye (such as Cy5, Alexa 647, Cy5.5, or Cy7) can be used with STORM. In this scheme, the activator fluorophore, when excited near its absorption maximum, serves to reactivate the photoswitchable dye to the fluorescent state. Multicolor imaging has been performed by using different activation wavelengths to distinguish dye-pairs, depending on the activator fluorophore used, or using spectrally distinct photoswitchable fluorophores, either with or without activator fluorophores. Photoswitchable fluorescent proteins can be used as well. Highly specific labeling of biological structures with photoswitchable probes has been achieved with antibody staining, direct conjugation of proteins, and genetic encoding. STORM has also been extended to three-dimensional imaging using optical astigmatism, in which the elliptical shape of the point spread function encodes the x, y, and z positions for samples up to several micrometers thick, and has been demonstrated in living cells. To date, the spatial resolution achieved by this technique is ~20 nm in the lateral dimensions and ~50 nm in the axial dimension; and the temporal resolution is as fast as 0.1–0.33s.


Points accumulation for imaging in nanoscale topography (PAINT)

Points accumulation for imaging in nanoscale topography (PAINT) is a single-molecule localization method that achieves stochastic single-molecule fluorescence by molecular adsorption/absorption and photobleaching/desorption. The first dye used was Nile red which is nonfluorescent in aqueous solution but fluorescent when inserted into a hydrophobic environment, such as micelles or living cell walls. Thus, the concentration of the dye is kept small, at the nanomolar level, so that the molecule's
sorption Sorption is a physical and chemical process by which one substance becomes attached to another. Specific cases of sorption are treated in the following articles: ; Absorption: "the incorporation of a substance in one state into another of a di ...
rate to the diffraction-limited area is in the millisecond region. The stochastic binding of single-dye molecules (probes) to an immobilized target can be spatially and temporally resolved under a typical widefield fluorescence microscope. Each dye is photobleached to return the field to a dark state, so the next dye can bind and be observed. The advantage of this method, compared to other stochastic methods, is that in addition to obtaining the super-resolved image of the fixed target, it can measure the dynamic binding kinetics of the diffusing probe molecules, in solution, to the target. Combining 3D super-resolution technique (e.g. the double-helix point spread function develop in Moerner's group), photo-activated dyes, power-dependent active intermittency, and points accumulation for imaging in nanoscale topography, SPRAIPAINT (SPRAI=Super resolution by PoweR-dependent Active Intermittency) can super-resolve live-cell walls. PAINT works by maintaining a balance between the dye adsorption/absorption and photobleaching/desorption rates. This balance can be estimated with statistical principles. The
adsorption Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...
or
absorption Absorption may refer to: Chemistry and biology *Absorption (biology), digestion **Absorption (small intestine) *Absorption (chemistry), diffusion of particles of gas or liquid into liquid or solid materials *Absorption (skin), a route by which s ...
rate of a dilute solute to a surface or interface in a gas or liquid solution can be calculated using
Fick's laws of diffusion Fick's laws of diffusion describe diffusion and were derived by Adolf Fick in 1855. They can be used to solve for the diffusion coefficient, . Fick's first law can be used to derive his second law which in turn is identical to the diffusion equ ...
. The photobleaching/desorption rate can be measured for a given solution condition and illumination power density. DNA-PAINT has been further extended to use regular dyes, where the dynamic binding and unbinding of a dye-labeled DNA probe to a fixed
DNA origami DNA origami is the nanoscale folding of DNA to create arbitrary two- and three-dimensional shapes at the nanoscale. The specificity of the interactions between complementary base pairs make DNA a useful construction material, through design of ...
is used to achieve stochastic single-molecule imaging. DNA-PAINT is no longer limited to environment-sensitive dyes and can measure both the adsorption and the desorption kinetics of the probes to the target. The method utilizes the camera blurring effect of moving dyes. When a regular dye is diffusing in the solution, its image on a typical CCD camera is blurred because of its relatively fast speed and the relatively long camera exposure time, contributing to the fluorescence background. However, when it binds to a fixed target, the dye stops moving; and clear input into the point spread function can be achieved. The term for this method is mbPAINT ("mb" standing for
motion blur Motion blur is the apparent streaking of moving objects in a photograph or a sequence of frames, such as a film or animation. It results when the image being recorded changes during the recording of a single exposure, due to rapid movement or lo ...
). When a
total internal reflection fluorescence microscope 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. TIRFM is an imaging modality which uses the excitation of fluorescent cel ...
(TIRF) is used for imaging, the excitation depth is limited to ~100 nm from the substrate, which further reduces the fluorescence background from the blurred dyes near the substrate and the background in the bulk solution. Very bright dyes can be used for mbPAINT which gives typical single-frame spatial resolutions of ~20 nm and single-molecule kinetic temporal resolutions of ~20 ms under relatively mild photoexcitation intensities, which is useful in studying molecular separation of single proteins. The temporal resolution has been further improved (20 times) using a rotational phase mask placed in the Fourier plane during data acquisition and resolving the distorted point spread function that contains temporal information. The method was named Super Temporal-Resolved Microscopy (STReM).


Label-free localization microscopy

Optical resolution of cellular structures in the range of about 50 nm can be achieved, even in label-free cells, using localization microscopy SPDM. By using two different laser wavelengths, SPDM reveals cellular objects which are not detectable under conventional fluorescence wide-field imaging conditions, beside making for a substantial resolution improvement of autofluorescent structures. As a control, the positions of the detected objects in the localization image match those in the bright-field image. Label-free superresolution microscopy has also been demonstrated using the fluctuations of a surface-enhanced Raman scattering signal on a highly uniform plasmonic metasurface.


Direct stochastical optical reconstruction microscopy (dSTORM)

dSTORM utilizes the photoswitching of a single fluorophore. In dSTORM, fluorophores are embedded in a reducing and oxidizing buffering system (ROXS) and fluorescence is excited. Sometimes, stochastically, the fluorophore will enter a triplet or some other dark state that is sensitive to the oxidation state of the buffer, from which they can be made to fluoresce, so that single molecule positions can be recorded. Development of the dSTORM method occurred at 3 independent laboratories at about the same time and was also called "reversible photobleaching microscopy" (RPM), "ground state depletion microscopy followed by individual molecule return" (GSDIM), as well as the now generally accepted moniker dSTORM.


Software for localization microscopy

Localization microscopy depends heavily on software that can precisely fit the point spread function (PSF) to millions of images of active fluorophores within a few minutes. Since the classical analysis methods and software suites used in the natural sciences are too slow to computationally solve these problems, often taking hours of computation for processing data measured in minutes, specialised software programs have been developed. Many of these localization software packages are open-source; they are listed at SMLM Software Benchmark. Once molecule positions have been determined, the locations need to be displayed and several algorithms for display have been developed.


Super-resolution optical fluctuation imaging (SOFI)

It is possible to circumvent the need for PSF fitting inherent in single molecule localization microscopy (SMLM) by directly computing the temporal autocorrelation of pixels. This technique is called super-resolution optical fluctuation imaging (SOFI) and has been shown to be more precise than SMLM when the density of concurrently active fluorophores is very high.


Omnipresent Localization Microscopy (OLM)

Omnipresent Localisation Microscopy (OLM) is an extension of Single Molecule Microscopy (SMLM) techniques that allow high-density single molecule imaging with an incoherent light source (such as a mercury-arc lamp) and a conventional epifluorescence microscope setup. A short burst of deep-blue excitation (with a 350-380 nm, instead of a 405 nm, laser) enables a prolonged reactivation of molecules, for a resolution of 90 nm on test specimens. Finally, correlative STED and SMLM imaging can be performed on the same biological sample using a simple imaging medium, which can provide a basis for a further enhanced resolution. These findings can democratize superresolution imaging and help any scientist to generate high-density single-molecule images even with a limited budget.


Combination of techniques


3D light microscopical nanosizing (LIMON) microscopy

Light MicrOscopical Nanosizing microscopy (3D LIMON) images, using the Vertico SMI microscope, are made possible by the combination of SMI and SPDM, whereby first the SMI, and then the SPDM, process is applied. The SMI process determines the center of particles and their spread in the direction of the microscope axis. While the center of particles/molecules can be determined with a precision of 1–2 nm, the spread around this point can be determined down to an axial diameter of approximately 30–40 nm. Subsequently, the
lateral Lateral is a geometric term of location which may refer to: Healthcare *Lateral (anatomy), an anatomical direction * Lateral cricoarytenoid muscle * Lateral release (surgery), a surgical procedure on the side of a kneecap Phonetics *Lateral co ...
position of the individual particle/molecule is determined using SPDM, achieving a precision of a few nanometers. As a biological application in the 3D dual color mode, the spatial arrangements of
Her2/neu Receptor tyrosine-protein kinase erbB-2 is a protein that in humans is encoded by the ''ERBB2'' gene. ERBB is abbreviated from erythroblastic oncogene B, a gene originally isolated from the avian genome. The human protein is also frequently refer ...
and
Her3 Receptor tyrosine-protein kinase erbB-3, also known as HER3 (human epidermal growth factor receptor 3), is a membrane bound protein that in humans is encoded by the ''ERBB3'' gene. ErbB3 is a member of the epidermal growth factor receptor (EGFR ...
clusters was achieved. The positions in all three directions of the protein clusters could be determined with an accuracy of about 25 nm.


Integrated correlative light and electron microscopy

Combining a super-resolution microscope with an electron microscope enables the visualization of contextual information, with the labelling provided by fluorescence markers. This overcomes the problem of the black backdrop that the researcher is left with when using only a light microscope. In an integrated system, the sample is measured by both microscopes simultaneously.


Enhancing of techniques using neural networks

Recently, owing to advancements in artificial intelligence computing, deep-learning neural networks ( GANs) have been used for super-resolution enhancing of optical-microscope photographic images, from 40x to 100x, from 20x with an optical microscope to 1500x, comparable to a scanning electron microscope, via a neural lens, and with positron-emission tomography and fluorescence microscopy.


See also

*
Multifocal plane microscopy Multifocal plane microscopy (MUM) or 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 differen ...
(MUM) *
Stimulated emission depletion microscope 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 ...
(STED) *
Photoactivated localization microscopy Photo-activated localization microscopy (PALM or FPALM) and stochastic optical reconstruction microscopy (STORM) are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging me ...
(PALM) *
Stochastic optical reconstruction microscopy 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 techni ...
(STORM) *
Deconvolution In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a deco ...
* Photoactivatable probes *
Correlative light-electron microscopy Correlative light-electron microscopy (CLEM) is the combination of an optical microscope - usually a fluorescence microscope - with an electron microscope. In an integrated CLEM system, the sample is imaged using an electron beam and an optical ligh ...
*
Super-resolution imaging Super-resolution imaging (SR) is a class of techniques that enhance (increase) the resolution of an imaging system. In optical SR the diffraction limit of systems is transcended, while in geometrical SR the resolution of digital imaging sensors ...
* Video super resolution


References


Further reading

* * {{Optical microscopy Microscopy German inventions Cell imaging Laboratory equipment Optical microscopy Fluorescence techniques