Editorial Feature

What is Nanoscopy? Techniques and Applications

The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan W. Hell and William E. Moerner for their breakthrough work in nanoscopy. Their work allows the field to be able to quite literally see past the photonic limits that govern optical microscopy. As new frontiers are opened at the atomic scale, the race is on to begin utilizing these discoveries in practical applications and techniques.

At the end of the nineteenth century two researchers by the names of Ernst Abbe and Lord Rayleigh discovered what was coined the “diffraction limit” for optical microscopy. The wave properties of light at the atomic level determined that beyond a certain size on the lateral and longitudinal planes of an object, roughly 250 nanometers (nm), that object becomes blurred and is unable to be resolved with modern optical technology. We could look at the cells, but we could not look into the cells at their atomic levels.

Electron Nanoscopy

Nanoscopy describes the ability to see past the generally accepted optical limit of 200 – 300 nm. The first example of nanoscopy came in 1933, when German Physicist Ernst Ruska developed an electron microscope that had a resolution which exceeded that which could be observed with an optical light microscope. While electron microscopes allowed researchers to see past the diffraction limit, they carried their own limitations.

Initial versions of electron microscopy made it difficult to observe a living cell in real-time at the atomic level. X-ray crystallography, for instance, resulted in the sample often being chemically fixed, dehydrated, and observed outside of the natural environment. We could look at the cell at its molecular level, but the cell was likely dead and we were unable to observe it in its living state, real-time.

Eventually advancements were made in electron microscopy that allowed researchers to observe molecules in their natural environment. Currently, researchers at Maastricht University in The Netherlands use high-resolution cryo-electron microscopy to create three dimensional images of specific proteins.

While this method is an improvement, it still requires delicate sample preparation and cannot allow for an interpretation of structures at the atomic level to the extent that newer techniques can.

Multiphoton Nanoscopy

The next breakthrough in nanoscopy came in the form of far-field and near-field multiphoton microscopy. Both of these methods allow for the spectra of individual molecules to be observed.

Far-Field – One method of far-field multiphoton imaging is called structured illumination microscopy, which uses interference between two laser beams to cause light patterns that reveal certain measurable spatial details.

Near-Field – Near-field scanning optical microscopy is able to pass the diffraction limit by illuminating a surface with a light being emitted from a very narrow aperture. Depending on the properties of the aperture used, samples can be resolved at the molecular level.

Both of these techniques provide the technical ability to observe objects in their natural environment at the atomic level, however they are very technically cumbersome to implement and their practical applications remain limited.

Super-Resolved Fluorescence Nanoscopy

The new frontier of atomic scale microscopy lies in super-resolved fluorescence. Indeed, it was these methods that resulted in the Nobel Prize. Both techniques utilize the properties of fluorophores, or fluorescent molecules, to illuminate objects at the atomic level. There are two primary methods to super-resolved fluorescence microscopy, ensemble and single:


The first method, called stimulated emission depletion (STED), was developed by Stefan Hell’s research group at the Max Planck Institute of Biophysical Chemistry in Göttingen, and utilizes the ensemble method of fluorophore microscopy.

This technique uses two laser beams to affect fluorophores in a particular nano-sized region of the sample. The first laser is used to excite the fluorophores and cause them to drop to a lower energy level, while the second, lower-wavelength beam causes the molecules to decay. Those molecules not excited remain in place and emit a fluorescent photon, allowing an image to be built. Fluorescence giveth’ as fluorescence is taketh’ away.


The second method recognized by the Nobel Committee involved work done by Eric Betzig and William Moerner on single fluorophore detection. The first observation of a single fluorophore happened in 1989 under the direction of Moerner and a fellow researcher.

They were able to measure the absorption spectra of a single fluorophore molecule, which represented a considerable breakthrough because now the molecules could be detected by their light emission and absorption.

It was this work that led to a number of methods for observing single fluorophore molecules, including stochastic optical reconstruction microscopy (STORM) and points accumulation for imaging in nanoscale topography (PAINT). Both methods allow the observer to optically move fluorescent proteins from active to inactive states.

This provides the ability to unlock the full potential of the natural photochemistry of a fluorescent protein and allows the observer to manipulate the proteins and build a three-dimensional picture of a section the sample.

Practical Applications of Nanoscopy

Super-resolved fluorescence microscopy is still a relatively new field, though recent discoveries continue to revolutionize it. The ensemble, or STED, method began having practical applications in 2000 and the single based methods in 2006. Already applications are breaking new ground in multiple areas of molecular biology.

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.

Additionally, as we discover novel new nanotechnological means to fight disease and repair biological systems, advances in nanoscopy will allow us to observe these new technologies in their natural environment and provide for diagnostic and practical medical breakthroughs only limited by the imagination.

Sources and Further Reading

Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.


Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Bessette, Will. (2019, September 12). What is Nanoscopy? Techniques and Applications. AZoNano. Retrieved on April 16, 2024 from https://www.azonano.com/article.aspx?ArticleID=3993.

  • MLA

    Bessette, Will. "What is Nanoscopy? Techniques and Applications". AZoNano. 16 April 2024. <https://www.azonano.com/article.aspx?ArticleID=3993>.

  • Chicago

    Bessette, Will. "What is Nanoscopy? Techniques and Applications". AZoNano. https://www.azonano.com/article.aspx?ArticleID=3993. (accessed April 16, 2024).

  • Harvard

    Bessette, Will. 2019. What is Nanoscopy? Techniques and Applications. AZoNano, viewed 16 April 2024, https://www.azonano.com/article.aspx?ArticleID=3993.

Tell Us What You Think

Do you have a review, update or anything you would like to add to this article?

Leave your feedback
Your comment type

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.