Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Picture a cell biologist hunched over a microscope, straining to make out what exactly is in the field of view. Are those two proteins on the inside of the cell membrane or on the outside? Or do they sit on opposite sides of the membrane?
Using most microscopes, those questions would remain unanswered.
Even top-of-the-line confocal instruments cannot distinguish the different possibilities, because cellular membranes, measuring just a few nanometers thick, fall far below their ability to resolve them clearly.
Whatever their configuration, if the proteins were closer than about 200 nm or so, they would appear to the viewer as a single blob.
Super-resolution microscopes, though, are another story. Depending on the particular method used, these microscopy systems can resolve objects spaced from 140 nm to about 20 nm apart. Applied to the proper sample, these techniques can be likened to a visually impaired individual trying on that first pair of glasses.
In one 2012 study, for instance, researchers led by Markus Sauer at Julius Maximilian University in Würzburg, Germany, used one such method to study the architecture of the Xenopus nuclear pore complex. With traditional widefield imaging, using fluorescently labeled reagents, the pores appear as a diffuse red fog. But when a super-resolution technique called dSTORM was applied, that fog resolved into discrete rings with a beautiful, eight-fold symmetry [1].
Initially, such microscopes were the exclusive toys of microscopy developers—researchers with the physical, optical and engineering expertise to design, build and troubleshoot precision, custom instruments. Today, they are available as turnkey products, many of which we reviewed in an article published last year. Here, Biocompare reviews some of the latest innovations in the super-resolution microscopy marketplace.
Carl Zeiss Microscopy
Though Zeiss offers systems for both photo-activated localization microscopy (PALM) and structured illumination microscopy (SIM) super-resolution imaging (ELYRA P.1 and ELYRA S.1, respectively), the company’s biggest development over the past few years has been the launch of the Airyscan detector for its LSM 800 and LSM 880 scanning confocal microscopes.
According to Joseph Huff, North American product marketing manager for laser scanning microscopy, the detector enables researchers to achieve super-resolution imaging throughout a complete 3D volume by effectively eliminating the pinhole that drives confocal microscopy and instead projecting 1.25 “Airy disks” onto a 32-element detector array. An Airy disk is the central, bright, circular region of the pattern produced by light diffracted when passing through a small, circular aperture.
For years, Huff explains, researchers have understood that confocal microscopes could boost their resolution by making the pinhole—which filters out out-of-focus light—smaller. But in doing so, signal-to-noise drops, thereby complicating imaging results. By combining the Airyscan detector with computational deconvolution, the system can improve resolution 1.7-fold, Huff says, down to 140 nm.
Though Airyscan is not new, the company will on May 3 launch a higher-speed implementation, which works by using an elongated excitation beam to scan four lines at once. “Typically, you raster a single point to build up a field of view,” Huff says. “Here, you collect four lines simultaneously.” This so-called “fast mode” will be available as a field upgrade on existing LSM 880 systems, he adds.
Leica Microsystems
Leica Microsystems supports two super-resolution methods, stimulated emission depletion (STED) and ground-state depletion followed by individual molecule return (GSDIM) microscopy.
According to product manager Peter Laskey, Leica’s GSDIM system (the Leica SR GSD 3D), built on a DMI8 inverted microscope scaffold, has in the past year been upgraded to a much faster camera that uses scientific complementary metal-oxide semiconductor (sCMOS) technology.
“The main advantage of the sCMOS is [that] by significantly accelerating data acquisition, we are able to get data in much shorter time frames, which enables you to get much greater throughput,” Laskey says. Using sCMOS, he explains, data acquisition can be accelerated from as much as 20 minutes per image to “well below a minute.” Another advantage: Faster acquisition means the sample is “less exposed to drift, so the data quality can also be said to improve.”
According to Robert LaBelle, vice president of marketing at Photometrics, a company that develops and manufactures cameras for the microscopy market, the challenge with super resolution is that each super resolved image is reconstructed from many individual frames, which is extremely challenging when imaging live cells. “You need cameras capable of very high frame rates and yet able to image just a few dozen photons per pixel in that period,” he says.
Most systems use one of two designs: electron-multiplying CCD (EMCCD) cameras or sCMOS cameras. The former offer superior sensitivity but cost “from the low-$20,000s to the mid-$30,000s,” according to LaBelle; sCMOS cameras trail slightly in sensitivity, but cost between $9,000 to $22,000. At the moment, LaBelle recommends EMCCD cameras (such as Photometrics’ Evolve® 512 Delta) for ultrahigh sensitivity and sCMOS (the Photometrics Prime) for “more mainstream imaging” applications. But, he notes, “sCMOS keeps improving every year. In the near future, say the next 12-24 months, I would predict that sCMOS will completely take over.”
Leica’s newest STED system, the Leica TCS SP8 STED 3X, launched in 2014, says product manager Jochen Sieber. Effectively an add-on module for the company’s SP8 confocal microscope platform, the STED 3X, which features three STED lasers and a white-light excitation laser, enables 3D super-resolution imaging “all over the visible range.”
Nikon Instruments
Nikon Instruments has updated its super-resolution product line with two new entries, the N-STORM 4.0 and the N-SIM E.
According to Stephen Ross, general manager of Nikon’s product and marketing department, the N-STORM 4.0 is essentially a live-cell-compatible version of the company’s original N-STORM microscope, with a magnification lens to concentrate excitation energy over a smaller field of view and a new sCMOS camera for faster data collection. “You can do all the things you could do with our previous STORM systems in a larger field of view,” he says. “But also, if you wanted to do what people would call live-cell STORM or single-particle tracking, you can do that with this system, as well.”
The N-SIM E is a “scaled-down,” lower-cost version of Nikon’s original structured illumination microscopy system, the N-SIM. N-SIM, Ross explains, costs about $600,000, putting it out of reach for many microscopy laboratories. With N-SIM E, the company eliminated certain imaging modes (such as total internal reflection SIM) in exchange for the $300,000 price tag. “It is really a personal structured illumination system,” Ross says. The system is also available as a combination SIM/confocal system, called the A1ER.
Olympus
Olympus’ newly launched SD-OSR super-resolution microscope is a high-speed SIM system implemented on a spinning-disk confocal microscopy platform.
Launched in November 2015, the SD-OSR comprises an IX83 inverted microscope outfitted with a Yokagawa W1 spinning disk unit, plus a magnifying lens that projects imaged Airy disks onto a detector, says product manager Russell Ulbrich. By spreading those Airy disks over multiple pixels and averaging multiple exposures per point, the system can acquire key “high-frequency” data, from which it can generate a high-resolution image.
Key to the system, Ulbrich says, is “a very low-noise environment.” Among other things, the SD-OSR uses a silicone-based immersion oil and matching objective to reduce spherical aberration. In addition, a correction collar lets users tune their optics to obtain the brightest possible images and hence make the most of that otherwise dim high-frequency information. “You tune it up for maximal brightness and signal,” he explains.
In total, the SD-OSR can achieve 120-nm resolution at up to three images per second, says Ulbrich, making the system particularly amenable to live-cell applications.
Other developments
Bruker has launched its first super-resolution instrument since its 2014 acquisition of Vutara Inc. Bruker could not be reached for comment, but according to a press release, the Vutara™ 352 “leverages high-performance data acquisition and image processing capabilities to perform the entire imaging workflow, from acquisition through localization to quantitative analysis.” When combined with the Opterra SR confocal scanner, the system purportedly can perform “seamless correlative imaging” to, among other things, “combine super-resolution images with confocal images for contextual information.”
Another development: In December 2015, Miltenyi Biotec launched a service in collaboration with Abberior, a company that develops fluorophores for super-resolution microscopy, to conjugate Abberior’s STAR dyes with primary MACS® antibodies. “Across the visible spectrum, nine dyes are available, which can be coupled to any of the company’s 900-plus monoclonal and recombinant antibodies,” says Miltenyi Biotec product manager Johannes Fleischer. Alternatively, users can submit their own antibodies for custom conjugation work.
According to Fleischer, STAR dyes are designed specifically for STED and will not work with localization methods such as PALM and STORM. (Abberior sells dyes for those methods, too, but they are only available from the company itself.)
With so many tools available, does super-resolution spell the end for traditional fluorescence microscopy? No, says Laskey, because “for many applications, the resolution that’s obtainable with a regular microscope is sufficient.” But, he adds, when the need strikes, there’s much researchers can learn from zooming in to super-resolution. “It’s fair to say [super-resolution microscopes] almost have now become relatively standard pieces of equipment in high-end microscopy labs.”
Reference
[1] Löschberger, A, et al., “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” Journal of Cell Science, 125:570-5, 2012. [PMID: 22389396]