super resolution 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 techniques rely on the near-field (photon-tunneling microscopy as well as those that utilize 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 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, 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 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 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 $\Omega$ 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 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 microscopy could potentially replace electron microscopy 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. 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