STEREO IN STEREO?
Courtesy of Microscopy U
Stereo microscopes come in two configurations, Greenough and the common main objective (CMO) designs. The more-expensive CMO design uses a single objective, whereas Greenough microscopes use two.
Stereomicroscopes are not sexy. They lack the power of modern compound instruments, the novelty value of a confocal, and the imposing presence of an electron microscope. They are routinely put to such unglamorous tasks as specimen sorting and dissection. But life scientists are now asking more of these faithful old workhorses, and the big four manufacturers – Zeiss, Leica, Nikon, and Olympus – are rising to the challenge, producing increasingly higher-resolution instruments with fluorescence and imaging capabilities that might even turn a few heads.
"Over the past 15 years or so stereomicroscopy has really started taking some big steps forward as far as performance goes," says Lon Nelson, Leica product manager. Aubrey Lambert, marketing manager at...
The dual light paths that give stereomicroscopes their name provide a three-dimensional image that, together with a high depth-of-field and a large workspace beneath the objective lens, permits access to the specimens with scalpels, needles, and other tools, and allows the user to judge where those tools are, relative to the specimen.
But these benefits come at the expense of resolution and magnification. "If you squash the depth of field down, then the microscope really becomes a compound microscope," says Nelson. Mike Metzger, a product manager at Nikon, says stereomicroscopes give way to high-power compound at a magnification "somewhere above 100×."
Leica currently offers the highest magnification, with a 16-to-1 zoom stereomicroscope with up to 115× standard magnification, with the option of supplementary lenses that can push that magnification to 200×. But, for routine manipulations such power isn't necessary. "Many of our microscopes are really ancient," says Mario de Bono, of the University of Cambridge's Laboratory of Molecular Biology, who uses them to sort worms for behavioral development studies. "They date from the 1960s, and they're still very good."
Stereomicroscopes are available in two basic configurations. The simplest is the "Greenough" design, which consists essentially of two low-resolution conventional microscopes angled in on the specimen. But, most biologists use a 'common main objective' (CMO) design (also known variously as 'telescope' or 'Galileo' due to the structure of the zoom optics), whereby two image paths arise from a single objective lens.
Because the two image paths in a CMO scope are parallel rather than diverging, additional components, such as extra lenses, fluorescence capabilities, and imaging equipment can be added in a modular fashion.
But you pay for that modularity. While a basic Greenough system (without illumination) such as the Olympus SZ 51 or Leica S6 might cost $1,500, CMOs start at around $3,500 and can run into the tens of thousands of dollars at the high end. "That's pretty expensive when you consider a regular tissue-culture or embryology-type inverted microscope would sell for three to five thousand dollars," says Nelson.
As a rule, high-quality optics, such as apochromatically corrected lenses (which align the whole of the visible spectrum in one plane), are the preserve of high-end CMO scopes. The exception is Leica's S8 APO. What this Greenough instrument lacks in modularity, it makes up for in optical quality, achieving higher resolution and double the magnification (80×) of other Greenough models. "It's a very good idea for people who want high optical quality on the cheap," says Michael Davidson, director of the Optical Microscopy Division of the National High Magnetic Field Laboratory at Florida State University.
For some jobs, such as fluorescence, the CMO scope's modularity is essential. "The key issue for us is sensitivity," says Daniel St. Johnston, principal research fellow at the University of Cambridge's Gurdon Institute, who uses fluorescence to detect protein traps in
Courtesy of Microscopy U
Adding fluorescence capabilities to a CMO microscope is a snap, given the design's modular architecture.
"Researchers are just continuing to push the boundaries with a broader range of fluorochromes, labeling fewer and fewer cells, maybe deeper and deeper within tissues," says Nelson. And fluorescence work is increasingly carried out on living specimens. "Illumination intensity means energy, and energy destroys living animals," says Gunnar Schroeder, product manager at Olympus-Europe. Low-intensity excitation is another driver for increased sensitivity, and has also led to the development of highly automated models such as Leica's MZ 16 FA, which illuminates in short bursts, enabling time-lapse image capture.
All of which pushes the total system cost higher. A full imaging system including a monitor and a sensitive digital camera might add an extra $15,000 to the instrument's cost. Meanwhile, different fluorochromes need different excitation and emission filters, which Schroeder says might cost $1,500 per set. "Most people use three of those," he adds.
Ergonomics is another advantage of CMO modularity. "In the standard biological lab, you have people who are 4'10" up to 6'5" working on the microscope," says Nelson. "Having the ability to tilt the binocular head is a big advantage."
In this respect, St. Johnston has a gripe with the high-end Leica models. "The eyepieces are just too high for any normal human being to use, and you have to spend another £1,000 on an adjustable head." St. Johnston has long used Leica scopes (he has about 10 in his lab) but is currently testing other makes and models.
St. Johnston's Cambridge colleague, Julie Ahringer, has had her Leica MZ FL111 for about a year, using it to sort fluorescently labeled worms. "At the time the Leica looked a bit brighter than the other ones," she says. In terms of optical quality, says Davidson, "Leica is definitely the Cadillac, but Zeiss' are catching up and will maybe even beat them."
Zeiss recently launched two new stereoscopes. Both its new flagship CMO-style Discovery microscope and its fluorescence-capable partner, the Lumar, are characterized by unusually large, high-numerical aperture objective lenses to achieve the highest possible resolution while maintaining depth-of-field. This has trimmed the working distance from 60 mm in previous models to 35 mm, but Lambert believes it is a price many biologists will be willing to pay. "If you've got a weak signal, you need a bright machine," he says.
St. Johnston, who has been comparing a loaned Lumar with a high-end Leica MZ16F, is impressed with the Zeiss: "We've looked at the things we always look at and we can see they're a bit brighter." But it doesn't come cheap: between £20,000 and £30,000, Lambert says. St. Johnston adds that the new Leica does not have the ergonomic drawbacks of its predecessors. He says the final decision on which instrument to buy might come down to which manufacturer offers the best deal.
At Cancer Research UK, Holger Gerhardt has also been purchasing new equipment for his laboratory, where he works on vascular development in mouse retinas. Among his acquisitions was an M2 Bio Quad, a rotating objective turret built by Kramer Scientific to fit a Zeiss SV11, enabling switching between stereo and high-power compound lenses. "We can have very nice overview pictures that enable us to look at the pattern," he says. "And then we can swap to high magnification to go to the individual cell level."
Adapting an SV11 (a discontinued model) in this way costs between $17,000 and $20,000, says Kramer's Neil Grossman. The company is developing a similar unit for Zeiss' Discovery. Leica provides an equivalent product in its Fluo Combi.
Gerhardt is now looking to take advantage of the trend for increasing motorization of such features as focus and zoom, which, via foot-pedal control, can leave both hands free for manipulations. He is especially interested in a motorized filter wheel. "If you have to fiddle around during image acquisition, you might offset your sample and the two images and the two different colors wouldn't really match up."
In opting for Zeiss equipment, Gerhardt admits he was swayed by more than optical quality and specifications. "Zeiss has a very good stand in our Institute. I've had experiences where I bought a new piece of equipment and the local support wasn't really good. That can be troublesome."
Manufactures will no doubt continue to trade off different components of microscope performance, to keep up with the needs of a dynamic discipline. But Nelson believes room remains for fundamental improvements, too. "As our manufacturing processes, and everyone's for that matter, improve, you should expect stereomicroscopes to get better and better," he says. "Of course, there is an eventual [physical] limit, but we are not there yet."