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Super-Resolution Microscopy – Get Your Free e-Book for Download

Until recently, the diffraction of light had placed a fundamental limit on how far biologists could peer into cells with optical microscopes, preventing them from resolving features less than 250 nm in size, missing critical structures within cells. Over the past 20 years scientists have developed several ingenious techniques allowing them to resolve features as small as 20 nm.

The Essential Knowledge Briefing, published by Wiley & Sons in partnership with Leica Microsystems, provides a general introduction to the field of super-resolution microscopy, describes potential problems and reveals forthcoming advances.

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Heath JP:
Super Resolution Microscopy

Essential Knowledge Briefing, published by Wiley & Sons

One of the first super-resolution techniques was stimulated emission depletion (STED) microscopy. It is based on a well-thought-out interplay of fine optics and photo physical processes and delivers super-resolution in a purely optical way on a confocal platform. Another technique called localization microscopy also stimulates individual fluorescent labels on a specimen, but it switches them on randomly rather than sequentially. There are different versions including photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and ground state depletion followed by individual molecule return (GSDIM). Structured illumination microscopy (SIM) relies more on computer processing and takes advantage of an interference pattern known as a moiré fringe. New super-resolution techniques that can achieve similar resolutions in the lateral and axial direction and thus provide 3D- images have begun to appear over the past few years (3D STORM, three-dimensional version of SIM, and isotropic STED).

Many steps in super resolution microscopy are similar to those in conventional fluorescence microscopy. However, the higher resolution means the labels need to be located much closer to each other to provide a detailed image. Presenting case studies, the EKB shows how scientists have already used super-resolution microscopy to image cellular organelles and structures that have never been viewed before with optical microscopy.

Super-resolution techniques come with the lack of speed: Certain features on an image of a live cell will appear blurred if they are moving on a timescale that is shorter than the imaging time. Also, the strong laser light can damage live cells. As most of the techniques use computer processing and various algorithms; there is a risk that these constructed images may contain artefacts.

A way to overcome some of the limitations is to combine super-resolution with other forms of microscopy. Combining fluorescence microscopy and electron microscopy (CLEM) allows the structures imaged by fluorescence microscopy to be placed within the landscape revealed by EM. Super-resolution microscopy enhances CLEM because its resolution is much closer to that of EM. To improve the tools they are working with, scientists have started to systematically assess the fluorescent labels for their ability to be used in super-resolution techniques.