In decades past, researchers and scientists have been constrained by an optical resolution limit—defined over 140 years ago—that established the maximum resolution of an optical system of 200 nm in the XY direction. This meant that many subcellular structures were not large enough for detailed observation, so biological research and science was limited.

This resolution barrier was shattered in 2014 when the Nobel Prize in Chemistry was awarded to three scientists “for the development of super-resolved fluorescence microscopy.” As a result, the microscope industry has seen a dramatic surge of interest in super-resolution microscopy techniques.

Super-resolution microscopy refers to any optical technique used to view samples at a higher resolution than the diffraction limit of conventional light microscopes. Today, scientists are using powerful super-resolution microscopy techniques to image living samples well beyond the established theoretical limit, allowing the study of far-ranging biological phenomena.

The primary benefit of super-resolution is enhanced resolving power, with several super-resolution options available that allow the imaging of structures below 200 nm. Some super-resolution techniques have allowed for improvements in resolution ranging up to an order of magnitude, bringing details into focus on a cellular or molecular level that had been invisible.

Other benefits of modern super-resolution microscopy include:

  • Observation at depth: Super-resolution microscopy allows the study of subcellular architecture and dynamics at the nanoscale. Researchers can clearly observe not only the surface of the sample, but also up to 100 µm deep within the sample.
  • Three-dimensional imaging: Researchers can obtain detailed three-dimensional super-resolution image data during time-lapse imaging thanks to higher temporal resolutions.
  • Ease of use: Some super-resolution microscopy techniques combine intrinsic optical sectioning with fast data acquisition and dual-color super-resolution to provide quality images in a timely fashion for further actions.

The limitations of super-resolution microscopes

These benefits appeared to be so promising that nearly every commercial microscope manufacturer began developing super-resolution microscopes. Today, these microscopes are almost as easy to use as a regular confocal microscope. They represent years of effort to create user-friendly super resolution microscopes that do not require experts to run and align them.

Initially, a wave of interest in super-resolution-enabled experiments swept through the research community. But interest waned as researchers learned how much effort was required to perform these experiments. The returns were not worth the efforts to modify sample preparation, alter imaging mediums, or perform tedious tasks like refractive index matching.

Widefield image of multiciliated cell basal bodies
Widefield image of multiciliated cell basal bodies made using the Olympus SD-OSR microscope system in super-resolution mode. [Hatsuho Kanoh, Elisa Herawati, and Sachiko Tsukita, Osaka University]

Lack of flexibility is another challenge encountered with many super-resolution systems—many hardware-based super-resolution systems cannot be easily adapted if an experimental protocol changes in the middle of an application. The quality of super-resolution images can also vary tremendously; images captured using the same resolution can appear dramatically different in detail and appearance.

Lastly, super-resolution systems are much more expensive than other high-resolution imaging options. A super-resolution system can start at more than twice the price of a confocal imaging system and can cost as much as four times more. The difference in cost between a super-resolution system and a wide-field fluorescence system is even more dramatic.

Can confocal microscopes perform super-resolution imaging?

It has been well established that reducing the pinhole size below 1 AU in confocal microscopes enables increased resolution. Theoretically, doing so could achieve the 2× increase in resolution that commonly defines super resolution. But in practice, it was found that resolution can be improved by only approximately 1.4× because high-frequency signals are weak when compared to plentiful low-frequency signals.

As a result, efforts focused on how existing technologies, like the confocal microscope, could be improved to use
existing high-frequency spatial information already present in these images. Naturally, deconvolution came to mind.

How does deconvolution work?

Deconvolution algorithms work by reassigning out-of-focus photons back to their original positions, based on a theoretical or acquired point spread function (PSF), to increase the sharpness and clarity of acquired images. In fact, deconvolution algorithms alone can reduce the size of a sub-resolution fluorescent bead full width at half maximum (FWHM) to achieve a measured 2x increase in resolution over wide field.

Let’s take a step back for a moment and define resolution in the context of microscopy. Traditionally, this is defined using the Rayleigh criterion, which specifies that there must be at least a 26% decrease in intensity between two objects to properly define them as two separate objects (Figure 1).

Rayleigh criterion figure
Figure 1. In optical systems, adjacent peaks for point object are generally considered distinguishable if irradiance dips at least 26% between them. This somewhat arbitrary criterion is known as the Rayleigh criterion.

More commonly, this is referred to as two-point resolution. Slimming down the FWHM of a sub-resolution object through deconvolution provides sharper images, but this may not be enough to achieve super resolution.

This begs the question: Is deconvolution enough to provide true super resolution data in actual samples? The answer is no.

One of the famous linear deconvolution methods is the Wiener filter. The Wiener filter handles all high-frequency data in the same way, which leads to the formation of ringing artifacts (Figure 2).

Image-enhancing algorithms simulated results figure
Figure 2. Image-enhancing algorithms may introduce ringing effects, or ripple-like artifacts near sharp edges. If sophisticated algorithms are used, the effects may be reduced, as illustrated with these simulated results: (A) Original object. (B) Confocal fluorescence microscope image. (C) Image after deconvolution with Olympus Super Resolution. (D) Image after linear deconvolution with a Wiener filter.

Some may argue that these artifacts are minimized by modifying the strength of the filter or locking researchers out of them completely. But the reality is, large artifacts are not acceptable when observing structures below the resolution limit.

More specific algorithms, such as Olympus Super Resolution (OSR), are designed to specially handle high-frequency data to provide more usable data, providing more resolution with fewer artifacts. These algorithms present a more comprehensive approach to super-resolution microscopy, making it simpler and more accessible than ever before.

Looking forward to the next 15 years

The world of biological microscopy is based on the ongoing effort to see further and further into what cannot be seen by natural means—the most subtle details and processes that define life. Super-resolution, one of the newest tools available to researchers, provides a highly advanced means to see even deeper, and more clearly, into the dynamics of living cells.

Great strides have been made in the world of super-resolution microscopy over the past 15 years, and many effective super-resolution techniques have become available. Nonetheless, super-resolution has not yet solidified its place as a primary tool for advanced imaging. Further advances in super-resolution will likely allow researchers to image even smaller structures for longer periods of time, and delve even deeper into living tissue.

With this new level of imaging support, experiments that once seemed out of the question are now a possibility. Fewer experimental sacrifices need to be made, and existing fluorophore labeling protocols can be used to integrate super-resolution technologies into workflows without disruption. The future is bright for microscopy techniques that have the capability of advancing with the technology of today and tomorrow.

 

Lauren Alvarenga is product manager, scientific solutions group, Olympus Corporation of the Americas.

This site uses Akismet to reduce spam. Learn how your comment data is processed.