In microscopy, one important ongoing objective is the desire to see smaller things more clearly. Conventional light microscopes face a resolution limit of around 200–250 nanometers, because of the diffraction limit of light. To see smaller features of a sample, scientists can apply super-resolution microscopy, which can reveal details that are just tens of microns in size. These novel technologies for imaging a sample provide resolutions that once seemed too good to be true—breaking a seemingly unbreakable barrier. Scientists must consider the advantages and disadvantages of these methods, and then find the one that provides the best fit to a particular research objective.
In general, super-resolution microscopy includes a few different kinds of technologies, such as stimulated emission depletion microscopy (STED). Another super-resolution method is single-molecule localization microscopy, which includes photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). Some scientists would also add structured illumination microscopy (SIM) to the list of super-resolution methods, but others would say that this technology cannot image much better than confocal microscopy.
This article will provide a brief explanation of how these technologies work. In STED microscopes, lasers turn fluorescent markers on and off in a very limited area, or focal point. Then, these lasers scan the sample to produce an image. In some cases, STED can be combined with live-cell imaging. With PALM and STORM, too, the fluorophores do not all get activated at the same time. In this way, the florescent markers get turned on in a sequence that can be used to build an extremely high-resolution image. Neither PALM nor STORM, though, makes a good choice for live-cell imaging, but these methods can be turned into platforms relatively easily. SIM technologies create a grid-like pattern of light that is shifted over an image, and then Fourier transforms are used to produce an image.
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Given that this technology is named super-resolution, just how good is it? The short answer is: better than ordinary light microscopy. So, that means that super-resolution microscopy must provide images that can resolve details smaller than 200 nanometers. But, some methods do that better than others. SIM takes the resolution to about 100 nanometers, which is about twice as good as an ordinary light microscope. With STED, some platforms provide 30-nanometer resolution with live cells, and 15 if the cells are fixed. With PALM or STORM, scientists can see details that are only about 15 nanometers across.
Better imaging is not only about better resolution.
As already pointed out, better imaging is not only about better resolution. The goal is seeing smaller things and being able to describe and interpret what they are. So, a super-resolution microscope’s signal-to-noise ratio also matters. A higher signal-to-noise ratio creates a clearer image, and that can matter the most. The specific comparisons depend on the particular platforms being used, the sample, and the imaging conditions, but the difference can be significant. For example, Ian Dobbie, manager of the advanced imaging unit at the University of Oxford, and his colleagues showed that SIM produced a signal-to-noise that was more than 900 times higher than STED’s in one comparison. For a scientist to know what signal-t0-noise will be possible, sometimes it just takes comparing a couple or more methods.
Timing considerations also come into play. If a scientists needs to see how something moves, the best choice is, well, again it depends. If something’s big, SIM can pretty quickly track movements, but it doesn’t provide enough resolution to do that on molecules. STED can be used in tracking, and the speed depends on the size of the image, because STED scans it. PALM and STED could also track dynamic events, but not very quickly, because these methods combine thousands of images into one.
Beyond the fundamental techniques, scientists keep expanding the capabilities of super-resolution microscopy, and here are a couple recent examples.
Modifying the methods
To really understand how biological systems work, the components must be viewed in context, and that can require three-dimensional (3D) imaging. Some techniques provide 3D imaging at high resolution, but not very deep into a sample. So, Pierre Bon, a researcher at the Laboratoire Photonique Numérique et Nanosciences, and his colleagues developed a method called SELFI, which they described as “a framework for 3D single-molecule localization within multicellular specimens and tissues.” Using this technology, the scientists imaged actin filaments down to 50 micrometers below a sample’s surface. In this work, the scientists used spheroids—3D cultures of cells—and the material didn’t even need to be cleared for imaging at these depths.
As mentioned above, imaging in space and time can be required in some applications. That can be done in various ways, and new methods keep emerging. Recently, Theo Lasser, head of the laboratory of biomedical optics at Ecole Polytechnique Fédérale de Lausanne, built a platform that combines 3D super-resolution microscopy and 3D phase imaging, which can be used to map cells and even organelles, and the scientists call this a 4D microscope.
Instead of building a super-resolution system, scientists can purchase one. For example, Nikon manufactures its N-SIM Super Resolution Microscope, which can be used for live-cell imaging and captures 15 frames per second. From Olympus, scientists can purchase the IXplore SpinSR10 super resolution imaging system, which was designed for live-cell imaging and provides 120-nanometer resolution. Both the Nikon and Olympus systems can be switched between different forms of imaging. For example, Olympus literature states that the IXplore SpinSR10 can “switch between super resolution, confocal, and widefield imaging.” To explore multiple details, scientists can select the HyVolution 2 from Leica Microsystems, which delivers 140-nanometer resolution and images multiple fluorophores. A super-resolution system doesn’t even need to take up a lot of lab space. For example, GE Healthcare describes its DeltaVision OMX SR as “a compact imaging system specifically optimized to provide a stable platform for structured illumination microscopy (SIM) super resolution technology,” and it can be used in several modes for live-cell imaging, including dynamic image capturing. That’s just a small selection of the available options.
The variety of decisions to be made and technologies to consider demands some research by a scientist before jumping into super-resolution microscopy, but the impact on research can make the time well-invested. The more details that scientists can discern in samples, the more we can learn about the world around us.