Super-Resolution Microscopy

True/Functional Techniques and Technology

Super-Resolution Microscope

Essentially, super-resolution microscopy (SRM) refers to any optical technique that is used for the purposes of resolving structures beyond the diffraction-limited resolution of conventional light microscopes.

The techniques of super-resolution microscopy allows for images to be viewed under higher resolution for fine mapping of such cellular structures as neural synapses and Golgi apparatus among others. 

Super-resolution can be achieved through a number of methods including:

  • 4Pi microscopy
  • structured illumination microscopy
  • stimulated emission depletion
  • reversible saturable optical fluorescence transition 
  • PALM

A Brief on Diffraction Limit 

Although a typical microscope allows for the visualization of the specimen by magnifying the image, ultimately it fails to see infinitely small details. This is largely due to the fact that such light microscopes are heavily limited by the fact that light is a wave that is subject to diffraction.

In a light microscope, the rays of light from the object converge onto a single point at the image plane during the imaging process. However, as a result of diffraction, the exact convergence of these rays is prevented which causes a sharp point on the object to blur in to a finite-sized spot on the image.

Whereas diffraction limit of resolution may not affect imaging at the organ or tissue level in light microscopy, it becomes a problem when zooming in to cells where a large number of the sub-cellular structures tend to be smaller than the wavelength. Here, it becomes difficult to use a light microscope to study cell structures in detail.

Super-resolution techniques overcome the challenges of diffraction limit making it possible to view and study the molecular structure of cells.

Super-resolution microscopy is broadly classified into two main categories. These include:

True Super-Resolution Techniques - This category of super- resolution microscopy captures information within the evanescent waves.

Functional Super-Resolution Techniques - This category of super-resolution microscopy employs experimental techniques for the reconstruction of the super-resolution image.

True Super-Resolution Techniques

True sub wavelength techniques include the techniques that use;

Near field scanning optical microscopy (NSOM)

Also referred to as scanning near-field optical microscopy, this technique uses fiber optic probe to funnel light to the nanometric dimension. Here, it becomes possible to take fluorescence measurements with a spatial resolution of tens of nanometers by scanning the probes near the surface of the sample. This is achieved by focusing the excitation laser through an aperture with a smaller diameter than the excitation wavelength, which results in an evanescent field on the other side of the aperture.

Through this technique, users obtain an order of magnitude improvement far above what can be achieved using a conventional fluorescent microscope. For instance, using NSOM, lateral resolution of 20 nm and vertical resolution of between 2 and 5 nm has been attained. Therefore, the technique proves unique and beneficial in that it helps bridge the gap between spacial resolution between the optical approaches commonly used and such high resolution techniques as electron microscopy.

One of the biggest advantage of ASOM is that it can provide both optical and spectroscopic data at high spatial resolution in combination with simultaneous topographic information.

It also has a number of limitations that include:

  • zero working distance and very small field depth
  • very long scan times for high resolution images/large specimen areas
  • very low transmissity of apertures smaller than the incident light wavelength

4Pi Microscope

4Pi-Microscope is a laser scanning fluorescence microscope that uses two high resolution objective lenses for illuminating the sample. Here, the sample is illuminated from both the back and front side.

Using this technique, the problem of blurred images is partially resolved since both focal light spots are superimposed, which helps remove the blur to a certain extent. Resolution largely depends on the effective 4Pi-spot, which is 3 to 5 times sharper than the spot of a regular confocal microscope. This allows the microscope to be used for studying various mitochondrial proteins and morphology.

Structured Illumination Microscopy Technologies

This microscopy technique uses a laser-based wide-field microscopy set-up in which a movable diffraction granting has been inserted into the excitation beam path. In this set-up, only the Zero order or first order diffracted laser beams are allowed to pass through the objective.

At the focal plane of the objective, these beams interfere with each other to create an illumination in stripe. Through superimposition with the sample, the stripe pattern generates a Moire effect. This technique offers significant advantages in that it enhances resolution beyond conventional resolution associated with diffraction, which in turn provides sufficiently high resolution to get dynamic images of live cells.

Using this technique, users can capture 85-100 nm distinction, which is very useful in biological sciences. Some of the benefits of SIM include:

  • 2x increase in spacial resolution compared to wide-field microscopy
  • 4D imaging at fast frame rate
  • Using this technique, there is no need to employ photoactivable or photoswitchable fluorophores.

While it has a number of advantages, it also has its limitations which include:

  • Artifacts may be generated during image reconstruction.
  • It is sensitive to out-of-focus light. This makes it difficult to use with thick/very densely labeled samples.

For most part, true super-resolution techniques are used in nano-science largely due to the fact that they provide super-resolution images directly. A good example of this is with NFOM which captures information contained in the evanescent wave close to the object.

Here, the images are obtained as long as the evanescent field around the object is directly measured. While these techniques have their strengths, super-resolution technique requires special equipment, which makes the techniques complex and hard to operate.

Functional Super-Resolution Microscopy

Functional super-resolution techniques include microscope techniques that use experimental techniques to reconstruct a super-resolution image.

This category is further divided into two groups:

Deterministic Super-Resolution - The techniques used here often use fluorophores, which is the most common emitter in biological microscopy. This shows a non-linear response to excitation that is used to enhance resolution

Stochastical Super-Resolution - These techniques are dependent on the chemical complexities of various molecular light sources that gives them a complex temporal behaviour. This factor makes several close-by fluorophores emit light at given separate times and become resolvable in time.

Deterministic Super-Resolution

Stimulated Emission Depletion Microscopy

Essentially, this technique is used to create super-resolution images through the selective deactivation of fluorophores and the minimization of the area of illumination at the focal point to enhance an achievable resolution for the system. 

Stimulated emission depletion microscopy (STED) microscopy uses two laser beams to illuminate the specimen. Here, an excitation laser pulse that is generally created by a multiphoton laser is followed closely by a doughnut-shaped red-shifted pulse (STED beam).

The excited fluorosphores that are exposed to this beam (STED beam) are instantaneously returned to the ground state by stimulated emission. Here, non-linear depletion of the fluorescent state by the STED beam is the basis for super-resolution. Although the two laser pulses may be diffraction-limited, the STED pulse in this microscope technique is modified in a manner that allows it to feature zero-intensity point at the center of focus with strong intensity at the periphery.

Once the two laser pulses are superimposed, it is only the molecules that reside at the center of the STED beam that are able to emit fluorescence. Through this action, the point spread function in effectively narrowed ultimately increasing resolution beyond the diffraction limit. In order to generate a complete image, the center zero is raster-scanned across the specimen in a similar manner to single-photon confocal microscopy.

In STED, the high the intensity of the STED beam, the more the molecules will emit a stimulated photon (than the spontaneous photon). STED microscopy offers 20nm of lateral resolution and 40 to 50 nm axial resolution. This makes it particularly useful in structural analysis, viewing life cells (when combined with other methods such as fluorescence correlation spectroscopy)

One of the main problems associated with STED microscopy is with regards to photo bleaching, which can occur as a result of excitation in higher excited states of excitation in the triplet state.

Ground State Depletion Microscopy

For this microscopy technique, fluorescent markers are typically used depending on given conditions. For instance, the marker may be freely excited from the ground state and return spontaneously through emission of the fluorescence photon. On the other hand, if light of appropriate wavelength is additionally applied, then the dye can be excited to a long lived state (where fluorescence does not occur).

Being a RESOLFT technique (REversible Saturable Optical Linear Fluorescence Transitions) ground state depletion exhibits a time-sequential readout from within the diffraction zone at given defined coordinates by using reversible saturable/photoswitchable transitions.

It also requires lower laser intensities employed in such techniques as STED given that it employs a metastable triplet state that has a lifetime range in the milliseconds to microseconds. With GSD microscopy, users will benefit from an acquisition time of between 2 and 10 minutes with a maximum resolution of 20 nm.

Saturated Structured Illumination Microscopy

Like SPEM (saturated pattern excitation microscopy) saturated structured illumination microscopy (SSIM) is a non-linear method that depletes the fluorophore ground state by saturated excitation thereby generating a sinusoidal emission pattern to be recorded onto the area-array CCD detector.

Here, the saturated excitation illumination produces narrow line-shaped regions on the zero nodes surrounded by high levels of fluorescence signal and generates a negative imprint of features that are being imaged.

Stochastical Super-Resolution

Photo-Activated Localization Microscopy

This technique is a form of localization microscopy that depends on the ability to isolate, detect and image individual fluorophores.

Nanometer scale resolution in this case is achieved by exploiting properties of photoswitchable fluorophores. Here, exposing the fluorophores to given wavelength brings about a change in emission spectra. Here, photoactivable and reversible photoswitchable fluorophores become fluorescent while the reversible photoswitchable fluorophores change from a fluorescent state to another.

With this technique, the basic principle is that low-power activating laser beam stochastically converts fluorophores with only a few of these being in their active state. The molecules are then imaged using a high-power illuminating laser beam so as to immediately convert then back to their inactive state.

This process (activation and inactivation) is repeated over thousands of frames in order to ensure that all the molecules have been imaged. With PALM, it is only by exciting the fluorophores stochastically in a controlled manner that each dot of fluorescence comes from one fluorophore.

With high labeling efficiencies and specimen with good signal to noise ratio, it is possible to achieve precisions down to 10 nm using this technique. 

Fluorescence Photo-Activated Localization Microscopy

Fluorescence Photoactivation Localization Microscopy (FPALM) brings together a number of existing technologies that produce images based on fluorescence of individual molecules. For this particular technique, lasers are used for the purpose of exciting dye molecules on the surface of the sample. Here, the lasers cause portions of the molecules to fluoresce giving off a light that creates the image to be captured digitally.

Like in PALM, this process is repeated with new sets of molecules being excited. The resolution of images using this technique has been shown to be about 20 times better compared to other traditional light microscopes. As such, the microscope is capable of creating images with a as low as 10 to 20 nm resolution.

Functional super-resolution techniques have become very important in biological imaging. This is largely due to the fact that they are relatively easy to realize as is the case with SSRM. Although they present various advantages, there are also a number of challenges with these techniques given that some techniques like STED may prove complicated with relatively slow image acquisition.

Super-Resolution VS Electron Microscopy 

There are a number of differences between super-resolution microscopy and electron microscopy. For instance, whereas super-resolution microscopy is a form of light microscopy (where light has to be manipulated to overcome the problems of light diffraction) electron microscopy is different in that it relies on electrons for imaging. On the other hand, super-resolution can be used to view and study cellular processes as they happen. This is to say that it can be used for viewing live cells and thus various cell structures or other molecules.

When it comes to electron microscopy, the sample is usually placed in a vacuum, which means that it is difficult to use electron microscopes to view and study live cells. Electron microscopy has an advantage over super-resolution microscopy, however, in that it is capable of much greater resolution that has allowed for better study of various molecular structures and protein molecules (EM spatial resolution approaches molecular and atomic levels). 

Super-resolution microscopy has been shown to be relatively easy to use as is the case with methods like structured illumination microscopy (SIM). Here, sample preparation has been shown to be more similar to such methods as wide field/confocal microscopy. This is not the case with electron microscopy, which requires more complicated procedures.

Applications and Benefits of Super-Resolution Microscopy

With super-resolution microscopy, scientists and researchers are no longer limited to the type of resolution that only relied on fixed tissue. Through such techniques as STED, it has now become possible to view and study live specimens and reorganization of dynamic protein. This is due to the fact that the technique can penetrate the tissue sample and allow for fast image acquisition.

While electron microscopy has a lot to offer in medical research and biology, it proves less beneficial when it comes to live specimen visualization in addition to the fact that electron microscopes are very costly with complicated specimen preparation procedures.  

Super-resolution microscopy also allows for detailed close-up images of such microbes as malaria parasites and viruses to be viewed and studied. As such, it overcomes the challenges of the typical microscopes making it a valuable tool in clinical research and biology. 

Some of the benefits of super-resolution microscopy include:

  • It can be used for studying sub-cellular architecture and dynamics at nano scale. This makes it particularly beneficial when it comes to studying cell structures.
  • Super-resolution microscopy combines intrinsic optic sectioning with fast data acquisition and dual color super-resolution to provide quality images in a timely fashion for further actions
  • It is capable of capturing molecules in actions, which can help study and understand their behaviour, interaction and other characteristics.
  • It is possible to carry out live cell imaging and colorization studies using super-resolution microscopy techniques.
  • Details that are smaller than 50 nm can be quickly resolved.
  • It can work with standard fluorophores.
  • It can minimize drift for accurate localization of molecules when combined with suppressed motion technology.

Related Articles:

Microscopy Imaging Techniques

Brightfield Microscopy - the most elementary microscopy technique but important to understand and apply correctly. 

Oil Immersion Microscopy - when used properly increases the refractive index of a sample/specimen. With only a few disadvantages, slides prepared with oil immersion techniques work best under higher magnification where oils increase refraction despite short focal lengths. 

The Confocal Microscope - check out how image details can be viewed through state of the art technology and lasers are impossible to view using a conventional microscope. 

Phase Contrast Microscope - learn about an entire new world that has opened up in the field of microscopy. Once limited to bright field illumination phase contrast observation is now a standard feature on almost all modern microscopes. 

Fluorescence Microscope - study the most used microscope in medical/biological fields which uses high powered light waves to provide unique image viewing options.

Dark Field Microscope - learn more about how when the light source is blocked off, light scatters as it hits the specimen and is then able to reveal details otherwise difficult to see. 

Polarizing Microscope - discover its use in a wide range of applications in fields such as geology, metallurgy and medicine. Essential in obtaining information about the color intensity, structure and composition of a sample.

Kohler Illumination - broaden your knowledge of this technique which evenly illuminates the viewing field providing a bright specimen image and eliminates glare. 

Differential Interference Contrast - a microscopy techique which benefits from differences in the light refraction by various sections of living cells and transparent specimens allowing for better visibility during microscopic imaging.

And how it all applies to Life Sciences.

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Wenhui Wang, Junnan Gu, Ting He, Yangbin Shen, Shaobo Xi, Lei Tian, Feifei Li, Haoyuan Li, Liuming Yan , and Xiaochun Zhou (2015) Optical super-resolution microscopy and its applications in nano-catalysis.




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