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 Beyond the diffraction limit - Super-resolution microscopy reveals amazing details With traditional light microscopy, the diffraction of light limits imaging resolution to about 250 nanometers. Super-resolution techniques can sometimes improve that by 10 times—or more
Bringing optical microscopy into the nanodimension!

A big honor for small objects: The Nobel Prize in Chemistry 2014 was jointly awarded to Eric Betzig, Stefan Hell, and William E. Moerner "for the development of super-resolved fluorescence microscopy!."

Today's microscopes provide a view of the unseen. Like no other invention, the microscope has revealed the secrets of nature. Ever since Robert Hooke first made his beautiful sketches of magnified insects, scientists have been peering at the world through microscopes. The microscopic world generally refers to things humans can't see with the naked eye. The human eye has a resolution in the order of 100 um (10-4 m), which is about the thickness of a hair. With the microscope, whole world become available, filled with knowledge that can serve as inspiration to our fantasy. But thanks to microscopes, scientists have the tools to visualize the detailed structures and dynamic processes inside living cells. Today's microscopes can reveal everything from the secretion of insulin in pancreatic cells to the chemical crossfire in slices of living brain tissue. Modern microscopes have come a long way since the days of Hooke and van Leeuwenhoek. "Nobody's looking with their eye anymore — everything's digital," [Source - said biophysicist David Piston of Vanderbilt University in Nashville, Tenn.]

For a long time optical microscopy (The optical microscope, is also known as light microscope, is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century.) was held back by a presumed limitation: that it would never obtain a better resolution than half the wavelength of light. Helped by fluorescent molecules the Nobel Laureates in Chemistry 2014 ingeniously circumvented this limitation. Their unprecedented work has brought optical microscopy into the nano dimension. In what has become known as nanoscopy, scientists visualize the pathways of individual molecules inside living cells. They can even see how molecules create synapses between nerve cells in the brain; they can track proteins involved in Parkinson's, Alzheimer's and Huntington's diseases as they aggregate; they follow individual proteins in fertilized eggs as these divide into embryos and lots more!! .

Today's microscopes provide a view of the unseen. Here, a false-colored scanning electron micrograph showing crystals of loperamide, which is a drug used to treat diarrhea.
How do microscopes improve our lives today?

The exploration of microscopes has led to numerous discoveries, without which we would be left with the limited knowledge our eyes give us. The advance of a scientific field often parallels the invention of instruments that extend human senses to new limits. A microscope lets the user see the tiniest parts of our world: microbes, small structures within larger objects and even the molecules that are the building blocks of all matter. The ability to see otherwise invisible things enrich our lives on many levels. Doctors can diagnose and treat diseases better, scientists are able to reveal links that help put criminals behind bars and make our world safer by examining the strength of bridges and other structures. Students also use microscopes to gain knowledge of the world around them.

The development of the standard microscope at the end of the 16th century would lead to a great step forward for science, particularly in biology and medicine. Thanks to these fascinating devices, man has been able to discover the wonders of the human body and study myriad ways to improve and protect it with medicinal treatments. The first scientific results based on microscopy dealt with the circulating blood system and changed our view of the human body. Scientists have also discovered and explored life's own building block – the cell. Different types of bacteria and the following struggle against diseases, as well as studies of different materials and their qualities are other valuable results.

From microscopy to nanoscopy. Optical microscopy is an invaluable tool for biophysical and biomedical research, particularly for studying living cells and organisms. Unfortunately, light diffraction limits the resolution of a lens-based (far-field) optical microscope to about half the wavelength of the incident light. To overcome this diffraction limit, researchers are developing "super-resolution" far-field optical microscopy. And in 2014, the Nobel Prize for Chemistry  was awarded to Eric Betzig, Stefan Hell and William E Moerner for their work in this field.
Super-Resolution fluorescence microscopy at the advanced bioimaging Core Researchers won a Nobel prize for giving microscopes much sharper vision than was thought possible, letting scientists peer into living cells with unprecedented detail to seek the roots of disease.
Light microscopy to Super resolution fluorescence microscopy

A simple microscope has one lens and is essentially a loupe or magnifying glass with a relatively high magnification. The basic modern microscope found in schools, hospitals, and research centers is a compound microscope which has a series of lenses to collect and focus the light transmitted through the specimen. Although larger and more complicated, the multiple lenses of the compound microscope increase magnification and resolution while reducing chromatic aberration. More sophisticated and specialized microscopes, such as an electron microscope, use the same scientific principles as their conventional counterparts even though they operate in a different manner. Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. Electron Microscopes were developed due to the limitations of Light Microscopes which are limited by the physics of light to 500x or 1000x magnification and a resolution of 0.2 micrometers.

In the early 1930's this theoretical limit had been reached and there was a scientific desire to see the fine details of the interior structures of organic cells (nucleus, mitochondria etc.). This required 10000x plus magnification which was just not possible using light microscopes. An electron microscope is a very large instrument that uses electromagnets for magnification and electrons for illumination. This remarkable instrument was developed by Knoll and Ruska of Germany in 1932 and it was put to use in 1940. It uses very high voltage electricity. Electron Microscope helps in observing sub cellular structures which cannot be seen by means of compound microscope. An internal vacuum is essential for its working. Magnification is 100,000 to 500,000.

A multitude number of technological developments and manufacturing breakthroughs over the past three centuries have led to significantly advanced microscope designs featuring dramatically improved image quality with minimal aberration. However, despite the computer-aided optical design and automated grinding methodology utilized to fabricate modern lens components, glass-based microscopes are still hampered by an ultimate limit in optical resolution that is imposed by the diffraction of visible light wavefronts as they pass through the circular aperture at the rear focal plane of the objective. As a result, the highest achievable point-to-point resolution that can be obtained with an optical microscope is governed by a fundamental set of physical laws that cannot be easily overcome by rational alternations in objective lens or aperture design. These resolution limitations are often referred to as the diffraction barrier, which restricts the ability of optical instruments to distinguish between two objects separated by a lateral distance less than approximately half the wavelength of light used to image the specimen.

Here comes the new microscopy and the term “super-resolution” that refers to methods that surpass the so-called diffraction limit. For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells. Applications are wide ranging – from dynamic vesicle movements to fluorescence images of sub-cellular structures, allowing researchers to see details in unprecedented detail.

This is the nucleus of a bone cancer cell. Using normal high resolution fluorescence microscopy, it is not possible to distinguish details of its structure. With super-resolved fluorescence microscopy, scientists can identify more than 100,000 proteins (appearing as different colors) altering the DNA structure of the bone cell. This view has an optical depth of about 600 nanometers. Super-resolved fluorescence microscopy was pioneered by chemists, physicists, and engineers—including three who won the 2014 Nobel Prize in chemistry. – [Source – National geographic].
Confocal (outer) and super-resolution (STED, inner) images of immunolabelled Titin Z-disk in sarcomers in fixed myofibroblasts. Only the super-resolved image discloses certain details of molecular organization. ---- Adapted from  Clausen et al NanoBioimaging
How Super-resolution fluorescence microscopy works?

Super-resolution fluorescence microscopy methods have overcome the longstanding diffraction barrier for far-field light-focusing optics and this techniques have the ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence. While electron microscopy allows identifying cell substructures until a resolution of ∼1 nm, the resolution of fluorescence microscopy is restricted to ∼200 nm to 250nm due to the diffraction limit of light. However, the advantage of this technique is the possibility to identify and co-localize specifically labeled structures and molecules. Among the various microscopy techniques, fluorescence microscopy is one of the most widely used because of its two principal advantages: Specific cellular components may be observed through molecule-specific labeling, and light microscopy allows the observation of structures inside a live sample in real time. Indeed, it was these methods that resulted in the Nobel Prize. This technique utilizes the properties of fluorophores, or fluorescent molecules, to illuminate objects at the atomic level. The ability to resolve molecules and watch processes at the nano-scale in real time allows for the ability to view the dynamics of molecular biological processes without harming the tissue itself. Advancements in medical imaging and treatment are sure to follow in the near future.

Hence, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. New advances in these techniques now give them the ability to image 3D structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale.  By applying multitude bar code–like excitation patterns in different orientations and processing all acquired images using computer algorithms, a high-resolution image of the underlying structure can be generated. These algorithms use the data (via various cross-correlation and minimization algorithms) to estimate the experimental parameters, such as the grating constant, phases and direction, unmix the multiple overlapping components in frequency space, and finally shift the moiré information back to the originating high frequency places to synthesize the image. With this approach the lateral resolution increases by a factor of two beyond the classical diffraction limit. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

 Alzheimer in Super-Resolution- Brain showing hallmarks of Alzheimer’s disease, plaques in blue. (Source: Zeiss) A new super-resolution imaging technique allows researchers to track how surface changes in proteins are related to neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Researchers have developed a new imaging technique that makes it possible to study why proteins associated with Alzheimer’s and Parkinson’s diseases may go from harmless to toxic. The technique uses multi-dimensional super-resolution imaging that makes it possible to observe changes in the surfaces of individual protein molecules as they clump together. The tool may allow researchers to pinpoint how proteins misfold and eventually become toxic to nerve cells in the brain, which could aid in the development of treatments for these devastating diseases. – [source - http://www.photonicsviews.com/alzheimer-in-super-resolution/]
Applications of Super resolution fluorescence microscopy

In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example,

  • video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution.
  • Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1–5 nm) resolution.
  • Investigations were performed on structures outside and inside of the nucleus, and on condensed chromosomes during cell division
  • Beyond imaging living or fixed cells, not just static images, which people often associate with microscopy, some scientists want to know how things move, which is possible through this microscopy.
  • Within medicine, for example, super-resolution microscopy is already providing insight into disorders associated with protein aggregation, such as Alzheimer's and Huntington's disease.

Thus, all the super resolution techniques have the potential of opening new avenues in biomedical research. Given the field is in nascent stage, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored. Also the uncertainties remain when considering the best choice of methodology. Discovery of new nanotechnological means to fight disease and repair biological systems will allow us to observe these technologies in their natural environment aiding in the proliferation of diagnostic and practical medical breakthroughs only limited by the imagination.

References

  • http://www.biocompare.com/Editorial-Articles/337535-Super-Resolution-Microscopy-Reveals-Amazing-Details/
  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835776/
  • https://cs371.stanford.edu/2017_papers/super_res_flouresence_imaging_single_molecules.pdf
  • http://jcb.rupress.org/content/190/2/165
  • http://iopscience.iop.org/article/10.1088/0022-3727/48/44/443001/pdf
  • http://www.photonicsviews.com/alzheimer-in-super-resolution/