A STED-y route to commercialization
2011; Future Science Ltd; Volume: 50; Issue: 6 Linguagem: Inglês
10.2144/000113682
ISSN1940-9818
Autores Tópico(s)Cell Image Analysis Techniques
ResumoBioTechniquesVol. 50, No. 6 Tech NewsOpen AccessA STED-y route to commercializationJeffrey M. PerkelJeffrey M. PerkelPublished Online:3 Apr 2018https://doi.org/10.2144/000113682AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken." With that blunt assessment, published in the Proceedings of the National Academy of Science in 2000, Stefan Hell introduced the world to a "super-resolution" microscopy technique called stimulated emission depletion, or STED.Or rather, he reintroduced the technique. Hell had first described STED—which cracks the diffraction limit that has for so long bounded optical microscopy—in a brief two-and-a-half page mathematical treatise six years earlier. "I had a very hard time to convince people it would work," Hell recalls. The idea of a "diffraction barrier," a physical constraint on light microscopy resolution, was simply too well ingrained.Following his 2000 article, though, researchers started paying attention; the publication has racked up some 310 citations, according to the ISI Web of Knowledge. It also attracted the notice of microscopy manufacturer Leica Microsystems who licensed STED technology in 2001 and began converting it into a viable commercial system. It would take some five years. Along the way, Leica's engineers, physicists, and microscopists, aided by members of Hell's own lab, would have to rethink most of the original benchtop design.Stefan Hell spent most of the 1990s -- and $30,000 of his own money -- formulating the idea for and constructing the first STED microscope.Courtesy of B. Schuller.The Seed of STEDIn the late 1980s Hell was a physics graduate student at the University of Heidelberg using confocal microscopy to measure features on computer microchips. "The actual thesis subject … was so technical that I started to think about more fundamental things that one could do with this outdated field of physics," he says, referring to optical microscopy. "I thought about breaking the diffraction barrier, which seemed to me a goal worthwhile to pursue scientifically."The diffraction barrier (also known as the Abbe limit) limits how closely together two objects in a cell can be and still be resolved using a light microscope. A function of the wavelength of light illuminating a sample and the physical properties of the optics used to visualize it, this barrier hovers around 200–250 nm, and it explains why microscopists cannot resolve macromolecular complexes such as densely packed microtubules into individual subunits via fluorescence light microscopy.Hell, though, had a gut feeling the barrier was less solid than imagined. He suspected it might be possible to overcome it by manipulating the "light-driven state transitions" of fluorescent dyes: in other words tinkering with the process by which the dyes absorb and release energy. He pursued the idea in the early 1990s, first at the European Molecular Biology Laboratory and then at the University of Turku in Finland, where he had a senior postdoctoral fellowship. In 1993, he had a breakthrough. "I realized that one could do it by turning off a dye by stimulated emission."Hell's vision was to use two superimposed laser spots, an excitation beam and a beam for molecularde-excitation, and alternate their pulses so that some of the molecules in the excitation area are prevented from fluorescing – the foundation of STED. "STED introduces a mechanism by which we keep molecules dark even though they are illuminated with excitation light," he says.As in standard confocal microscopy, the excitation beam excites the fluorophores in a diffraction-limited region in the sample, rasterizing across the sample and collecting fluorescence intensity spot-by-spot. First, though, the STED beam—shaped like a doughnut with a hole in the center—deactivates the dyes at the periphery of that relatively large spot by forcing them back down to their ground state without fluorescing, acting like a light-based photomask. The net result is to shrink the effective excitation region below the diffraction limit.Marcus Dyba, a Hell lab ex-patriot who helped Leica develop the TCS STED, with the original microscope.Courtesy of A. Fromann."The purpose of that ring is to keep molecules fluorescent at the center of the doughnut while at the periphery, where the [STED laser] intensity is strong, the molecules are shut off," Hell explains. "So the region in which molecules are allowed to emit is made smaller in the end, and so it's possible to see much finer detail."Hell published his idea in 1994 together with Jan Wichmann, a lab intern who helped with the numerical simulation. But as a postdoctoral researcher, Hell lacked the means to apply for grants and implement the idea, as well as cover the costs of patenting it. So, he turned to the Fraunhofer Society, a German scientific organization that, at the time, maintained an agency supporting freelance inventors. Fraunhofer provided Hell a stipend covering about 75% of the cost of applying for intellectual property protection in the US and the European Union. (The remaining 25%, about $30,000, he paid out of his own pocket.) In exchange, the Society retained the right to license the intellectual property itself.What he needed next was a lab in which to actually build his system, and the money to fund the work. The Max Planck Institute provided the former, offering Hell a position in 1997, and even kicked in some of the latter. But it wasn't until the German government ponied up some additional funding in 1998 that he had the resources necessary to begin construction. Finally, in 1999, he had a working system.Hell's first paper, published that year in Optical Letters, improved resolution from 150 nm to 106 nm—"clearly beyond the Abbe limit," he wrote, and sufficient to resolve two adjacent nanocrystals that could not be distinguished using standard confocal microscopy. The follow-up report, in PNAS in 2000 (both Science and Nature passed on the manuscript), improved the resolution still further to about 97 nm, and demonstrated the technique's applicability to the life sciences by imaging both yeast and E. coli. The overall improvement amounted to six-fold in the axial (z) direction, and two-fold laterally; subsequent improvements bumped the lateral resolution to six-fold as well. Today, the maximal resolution of STED microscopy is around 50 nm, on the order of the size of a ribosome.The Road to CommercializationLeica's decision to license STED did not come out of the blue. The company had a history with Hell, licensing an earlier super-resolution technique he had developed, called 4Pi."We knew that his ideas had the potential to be converted into products," says Tanjef Szellas, who heads Leica's confocal team and previously was STED product manager.Still, the road to STED commercialization wasn't easy. A photograph of Hell's original design, published in a 2006 report of the Max Planck Society entitled Wissen Hoch 12, bears little resemblance to a traditional microscope. Arrayed on an open optical bench measuring perhaps 1 m × 3 m are a series of mirrors, lenses, cables, and other assorted black boxes with silver fittings. A laser beam bounces from mirror to mirror, tracing a zigzag path across the bench. Conspicuously absent are the usual iconic microscope shape, oculars, objectives, and a stage.The challenge for Leica's engineers was to shrink and fold those components, like optical origami, into a box measuring 25 cm × 25 cm × 15 cm, which could be plugged into the company's existing scanning confocal systems.The core of the STED development team included just three or four people, says Hilmar Gugel, optics product design manager at Leica and the former STED project manager, though between ancillary players in software, electronics, and engineering, "probably 15 to 20 were involved all together." Included in that group were several Hell lab ex-pats, including Gugel and physicists Marcus Dyba and Arnold Giske. (Two other Hell lab alumni have also held positions at Leica, helping both with the 4Pi system and a new Hell-developed super-resolution system called ground state depletion and individual molecule return (GSDIM).) "The transfer of the knowledge was facilitated significantly by that," Szellas says.According to Szellas, one of the biggest engineering challenges was a direct consequence of the system's heightened resolution. Because standard microscopes are diffraction-limited, their components can be built using tolerances in the 100-nm range. "Any jitter of, say, 50 nm in the laser beam wouldn't be that easy to see with conventional resolution," he says. But on the other hand, "if you have a movement of the two beams in the STED system, you would see that immediately."Hell's team handles this problem the old-fashioned way: by finely adjusting mirrors. But this approach wouldn't work for a commercial system in a closed box, so Leica's engineers installed an autoalignment function. Every two hours or so (depending on environmental variables), users press a button that moves a reference object into the optical path (without removing the specimen under investigation). This object is then imaged using both the excitation and STED lasers, the system adjusting its mirrors until the two superimpose correctly.System components presented another challenge. For instance, the original design used a relatively large, expensive (about $250,000, according to Hell), high-energy Ti:sapphire laser as a STED beam. The excitation laser was also large. And both, in Hell's design, operated in the open. To shrink things down and make them safer for general use, the Leica team decoupled the lasers from the system with fiber optics, which had the dual benefit of enabling them to pack components more closely together and increasing system reliability. "If the distance the light travels is short, it is more stable," Gugel explains.Also requiring re-engineering was the phase filter, the component that shapes the STED beam into its characteristic torus. Hell's design employed a "programmable spatial light modulator," made of liquid crystal. As light bounces off this surface, explains Gugel, "it modifies the wavefront to make the doughnut. But [the device] is huge and also quite expensive, and so not an option for us." Instead, Gugel, an optical physicist, designed (and patented) a new phase filter, which exploits the wavelength changes light undergoes as it passes from air to glass to metal.Lessons LearnedAs Leica's engineers labored on STED, they relied on lessons learned from that earlier collaboration with Hell that produced the Leica TCS 4Pi. Though the company sold several units, according to Szellas, the 4Pi was expensive and challenging to use. It imaged samples jointly from above and below, and displayed images from the side (that is, in the x-z plane) rather than from above—something microscopists were not used to. It also employed mathematics to clean up and produce the final picture.In contrast, operation of a STED microscope is more or less the same as standard confocal microscopy, Szellas says. "What I learned from the 4Pi microscope is that it is very important that the microscope is very easy to use and stable," says Gugel, who also was involved in the development of the TCS 4Pi.Leica launched the TCS STED in 2007 with a price of about $1 million. The physical product, says Hell, was a "neatly engineered and compact" system bearing "most of the physical-technical ingredients that we had in the laboratory … [plus] a lot of software that made it very easy to use." In 2009, the company released a second system called the TCS STED CW. Whereas the original STED system requires dyes (such as ATTO-647) that emitted deep in the red end of the spectrum, the new system is compatible with more popular green fluorophores and fluorescent proteins, thereby simplifying live-cell imaging.Swapping the expensive Ti:sapphire laser for less-expensive continuous wave lasers and with a simplified setup, the CW version costs about half as much as the first-generation instrument, says Szellas. By the end of this year, he estimates several dozen CW systems will be installed worldwide, plus some 20 Ti:sapphire-based units.The Leica TCS STED CW confocal microscope including the laser rack with depletion laser and electronics (left), air dampened optical table with inverted microscope, scanning unit, and STED module.Right: Computer and supply unit with excitation lasers.For the end-users, the result of Leica's STED development was technology democratization. According to Szellas, the system made super-resolution "usable for the daily , research of life science researchers … rather than being accessible only for experienced biophysicists." One of those first-gen system adopters was Silvio Rizzoli, a group leader at the European Neuroscience Institute in Goettingen.Nuclear structures visualized with Chromeo 494 (green) a d At o 647N red using confocal left and STED (right) microscopy.Image courtesy of Dr. L. Schermelleh.Rizzoli, who has published 15 papers using STED, did his postdoctoral research at the Max Planck Instutute (though not in Hell's laboratory), and used the original STED installation. Or rather, he obtained data on the instrument—he never actually ran the system himself. "There was always a physicist that would place the sample on the microscope," Rizzoli recalls. "Sometimes it took weeks of setting up the scope to take a few pictures." That's because, as an open system, it required significant tweaking to align all the beams to make it work correctly. As a mere biologist, he says, "I didn't use it directly."Rizzoli's lab studies the biology of neural synaptic vesicles, and now, using a TCS STED instead, he can run his own experiments. "What we found was that when you fuse the vesicle to a membrane, the [vesicle cargo] molecules do not separate but stay together," he explains. "You cannot resolve these molecules in a confocal microscope; they appear as a clump. But in STED, you see clusters of synaptic molecules together." After a several-hundred-thousand-dollar upgrade to handle two-color experiments, the scope, says Rizzoli, is "quite a solid instrument," and also "much more versatile." As a full-featured confocal, it also supports applications such as two-photon microscopy.Of course, not every microscope born into an academic lab makes it so far, or follows the same path to commercialization. In 2009, Nikon announced two super-resolution microscope systems of its own, the N-SIM and the N-STORM. The former was based on a technology invented by Mats Gustaffson in the lab of John Sedat at the University of California, San Francisco; the latter stems from work by Xiaowei Zhuang's lab at Harvard University.According to general manager Stephen Ross of the product and marketing division at Nikon Instruments, though the two instruments were launched simultaneously, the N-STORM took only about a year to bring to market, whereas the N-SIM took five. That, he explains, is because the former required mostly a software upgrade to Nikon's existing TIRF-based systems, whereas the latter represented more significant hardware development. "For SIM, we had to design from the ground up," Ross says.In a PNAS editorial published two weeks after Hell's 2000 publication, Shimon Weiss of the Lawrence Berkeley National Laboratory wrote that the invention "has the potential to transform the fluorescence microscopy 'Renaissance' we are currently experiencing into an 'Enlightenment Millennium.'" Certainly, if citation counts and user uptake are any guide, STED is making traction.But for Hell, that vindication is not what counts. "I have a lot of fun showing you can do this more simply than anyone thought," he says.FiguresReferencesRelatedDetailsCited ByAn introduction to optical super-resolution microscopy for the adventurous biologist16 March 2018 | Methods and Applications in Fluorescence, Vol. 6, No. 2 Vol. 50, No. 6 STAY CONNECTED Metrics History Published online 3 April 2018 Published in print June 2011 Information© 2011 Author(s)PDF download
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