physics

Super-resolution microscopy

Super-resolution microscopy is a physics topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Super-resolution microscopy rather than just read about it. In short: 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…

Key takeaways

Super-resolution microscopy belongs to physics; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect Super-resolution microscopy to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of Super-resolution microscopy from memory before moving on to harder problems.

Reference excerpt

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 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.

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. Guerra. (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. patents 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 published 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.

… excerpt ends here. Continue reading the full article.

Illustrations

Worked examples

Example 1 — a first encounter with Super-resolution microscopy

Start with the simplest possible case. Write down what Super-resolution microscopy claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In physics, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Super-resolution microscopy before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Super-resolution microscopy ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Super-resolution microscopy

In research: Super-resolution microscopy appears in physics research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses Super-resolution microscopy in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: Super-resolution microscopy is common in secondary-school and first-year university syllabi. It links to neighbouring topics Cell imaging, Fluorescence techniques, German inventions, so understanding it makes those chapters shorter.
In everyday life: Look for Super-resolution microscopy outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study Super-resolution microscopy in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what Super-resolution microscopy means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain Super-resolution microscopy out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Super-resolution microscopy in simple terms?

Why does Super-resolution microscopy matter?

Because it connects several physics ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Super-resolution microscopy?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Super-resolution microscopy.