Super-resolution microscopy is a series of super-resolution imaging 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 (photon-tunneling microscopy as well as those that use the Pendry Superlens and near field scanning optical microscopy) or on the far-field. 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 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 SIMU.S. Pat. No. 5,666,197: Apparatus and methods employing phase control and analysis of evanescent illumination for imaging and metrology of subwavelength lateral surface topography; John M. Guerra, September 1997. Assigned to Polaroid Corp. 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, show a nonlinear response to excitation, which can be exploited to enhance resolution. Such methods include STED, GSD, RESOLFT 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 (SOFI) and all single-molecule localization methods (SMLM), such as SPDM, SPDMphymod, PALM, FPALM, STORM, and dSTORM.
On 8 October 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, W.E. Moerner and Stefan Hell for "the development of super-resolved fluorescence microscopy", which brings "optical microscopy 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 C. Cremer and T. Cremer (1978): Considerations on a laser-scanning-microscope with high resolution and depth of field Microscopica Acta VOL. 81 NUMBER 1 September, pp. 31—44 (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.The Nobel Prize in Chemistry 2014 https://www.nobelprize.org/prizes/chemistry/2014/hell/biographical/
Some of the following information was gathered (with permission) from a chemistry blog's review of sub-diffraction microscopy techniques.Part I and Part II
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 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...".
Super-resolution techniques
Photon tunneling microscopy (PTM) Photon tunneling microscopy (PTM) provides real-time whole-field non-scanning imaging with sub-wavelength lateral resolution and vertical resolution as low as 1 nanometer, as demonstrated by John M. GuerraGuerra was elected a member of the National Academy of Engineering in 2024 "For inventing and commercializing nano-optics to advance super-resolution microscopy and produce hydrogen directly with sunlight".. (Note the differentiation of PTM with photon scanning tunneling microscopy (PSTM), which is essentially a re-naming of near-field scanning optical microscopy (NSOM) that exploits the near-field from a sharp optical fiber tip positioned extremely close to the sample surface, typically within a few nanometers, such that when the fiber is scanned along the sample surface an image is built up).
PTM provides sub-wavelength spatial resolution due to its capture of bound high-frequency diffracted light at the sample and conversion into image-forming propagating light. Easy insertion of the sample surface into the evanescent field at the distal end of the microscope is provided by a flexible "transducer" of a polymer or thin glass that is placed proximal to the sample. This transducer is flexible enough to follow macro-curves on the sample while providing a stiff flat reference locally. The vertical resolution is limited only by the photometric resolution of the whole-field detector, and can be as low as 1 nanometer. The technique has been applied to biological and solid-state samples, offering insights into surface morphology and optical properties at the nanoscale.
Phase-shifted PTM aka Phase-shifted Evanescent field Microscopy (PEM) In further development of this work in 1996, John M. Guerra showed that super-resolved lateral topography even higher than is achieved with PTM is attained by phase-shifting the evanescent field. In propagating light, planes of equal phase are parallel to planes of equal amplitude, such that phase-shifting produces sub-wavelength super-resolution along the vertical axis, such as achieved with interferometers produced by WYKO and ZYGO for measurement of optical surfaces. However, the evanescent field is inhomogeneous in that the planes of equal amplitude are parallel to the sample surface (thus the high vertical resolution in PTM), while the planes of equal phase are perpendicular to them and to the surface. Shifting the phase therefore now can produce sub-wavelength super-resolution in the lateral axis that is even higher than that of PTM. While there are many ways to initiate this phase shift, as taught by Guerra in several U.S. patentsU.S. Patent Number 5,774,221, John M. Guerra, Apparatus and methods for providing phase-controlled evanescent illumination (1996); Number 5,666,197, John M. Guerra, Apparatus and methods employing phase control and analysis of evanescent for imaging and metrology of subwavelength lateral surface topography (1996), and Number 5,715,059, John M. Guerra, Dark field, photon tunneling systems and methods (1996) assigned to the Polaroid Corporation, the first demonstration employed a Meadowlark Optics phase retarder and polarizer combination. Subtracting consecutive images for which the phase was shifted by a small value produced resolution 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. In 2001, with Dmitri Vezenov, Guerra publishedNIST ATP Funding No. 70NANB7H3054: Calimetrics East Final MORE Report, December 15, 2001, page 5. a fully-resolved image of 200nm spheres, close-packed hexagonally, while illuminated with 650nm light, for a resolution of 0.3 lambda. While impressive, potential resolution can be much higher, as it is limited only by the magnitude of the phase-shift.
Local enhancement / ANSOM / optical nano-antennas Apertureless near-field scanning optical microscopy (ANSOM), also known as scattering-type scanning near-field optical microscopy (s-SNOM), achieves super-resolution imaging through the local enhancement of the optical field near a sharp tip, often made of metal. In ANSOM, a metallic or dielectric probe interacts with incident light, creating a confined and enhanced electromagnetic field at the apex of the tip due to localized surface plasmon resonance.
Optical nano-antennas can further improve resolution by acting as resonators that concentrate light into nanoscale volumes, enhancing local field strength and sensitivity. These nano-antennas are engineered to resonate at specific optical frequencies and are used both in illumination and detection modes to achieve spatial resolutions well below the diffraction limit.
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.US patent 2009/0116,024, priority date 7 April 2006: J. V. Mikliaev, S. A. Asselborn Method for obtaining a high resolution image
NORM uses 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 with an improved axial 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.
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 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 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 in fixed cells and RESOLFT 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 (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 could potentially replace electron microscopy as a tool for some medical diagnoses. These include diagnosis of kidney disorders, kidney cancer, and blood diseases.