Structured illumination microscopy (SIM), for example, in which the sample is illuminated with patterned light, can achieve twice the spatial resolution of conventional optical microscopy. Meanwhile, techniques such as STED (stimulated emission depletion) fluorescence microscopy, RESOLFT (reversible saturable optical fluorescence transitions), PALM/STORM (photo-activated localization microscopy/stochastic optical reconstruction microscopy) and SSIM (saturated structured illumination microscopy) push the limits of optical resolution to the nanometre scale.

"Super-resolution optical microscopy has been one of the most momentous developments in life science over the last decade," said Christian Eggeling, from the University of Oxford. "Still, a lot of confusion exists on what the various super-resolution techniques can really accomplish in biological research and what the upcoming challenges are." To address these uncertainties, Eggeling has helped put together "The 2015 super-resolution microscopy roadmap", published in the Journal of Physics D: Applied Physics (J. Phys. D: Appl. Phys. 48 443001).

Organized by Eggeling and Mark Bates from the Max Planck Institute for Biophysical Chemistry, the roadmap brings together 15 short review articles from experts in the field. Covering a wide range of techniques, the articles discuss the underlying concepts of super-resolution optical microscopy, the potential and drawbacks of the different approaches, and applications in which these methods will have a significant impact.

"I liked the idea of having a collection of short and precise comments from leading scientists in the field on this topic – this is much easier to read and much more versatile than a very long review article by one group," explained Eggeling.

Resolution revolution

In the first article of the collection, Stefan Hell and Steffen Sahl from the Max Planck Institute for Biophysical Chemistry describe the emergence of super-resolution microscopy, stemming from the realization that a major leap in spatial resolution can be achieved by exploiting the spectral properties of the imaged molecules – and not by fighting against the phenomenon of diffraction.

 "As a major paradigm shift, molecular states and the transitions between them have emerged as the enabling element and the key to radically overcoming the diffraction barrier in far-field optical imaging," they write.

Hell and Sahl note that in little more than a decade, early theoretical proposals have been realised as powerful imaging strategies. Far-field optical methods are now routinely used to probe transparent matter at length scales just a tiny fraction of the wavelength of the imaging light, and are starting to enable numerous discoveries in molecular biology, neuroscience and beyond. 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.

Looking ahead, Hell and Sahl point out that there is still much work to perform in this new field. In particular, there's a real need to move more towards real-time, four-dimensional molecular analysis, not only in cells, but also in tissue-like preparations or tissues themselves. Improvements in detectors and lasers will also be major drivers of technical improvement and cost reduction, and thus ease of applicability.

The roadmap also includes articles describing the development and use of techniques including STORM, SIM, CSREM (correlative super-resolution optical and electron microscopy) and RESOLFT. Other authors take a look at advanced approaches such as the use of adaptive optics for super-resolution microscopy, the production of DNA-based reference samples for microscope calibration, advanced image deconvolution methods for STED, and tailoring fluorescent probes to optimize super-resolution microscopy.

There's also an article discussing the use of fluorescent nanobodies, a novel way of labelling proteins with very small tags, for high-resolution imaging, plus a paper that presents examples of in vivo studies on mice and other animal models. Finally, several articles describe applications of super-resolution microscopy, including the imaging of synaptic structures for neuroscience studies, investigations of plasma membrane organization and immunological research.

"The timing of this publication is perfect, since the Nobel Prize in Chemistry was given to this topic just a year ago," Eggeling told medicalphysicsweb.

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