by Jeffrey M. Perkel
The eukaryotic cell has never looked so good. From the pages of scientific journals to the covers of biology textbooks, cells are being captured at their microscopic best, their tightly focused structures awash in shades of fluorescent blues and greens, reds and yellows.
For microscopy experts, capturing such images is just part of the job. But for newbies, it can be a frustrating exercise in experimental design, optical alignment, software navigation and basic technique.
Michael Davidson, a microscopy expert at the National High Magnetic Field Laboratory at Florida State University and developer of the microscopy education web site Molecular Expressions, says students in his lab often initially struggle with bringing images into focus. “If you are doing widefield microscopy at 40x or 60x, you can see a number of different planes, and they have a hell of a time figuring that out,” he says.
Scott Olenych, imaging product marketing manager for Carl Zeiss Microscopy, says his new clients often struggle with experimental design, especially concerning which dyes work well together and how to produce quantifiable data.
Tech support can help in these situations, but over the long term there is no substitute for experience. Microscopy, like mass spectrometry and PCR, is a tool, albeit an exceptionally powerful, versatile and perhaps artistically gratifying one. For those researchers who might wish to incorporate microscopy into their research but are intimidated by its apparent complexity, fear not. Vendors have developed an array of hardware and software tools to help.
A confocal-in-a-box
One approach to simplified microscopy is to treat the microscope as a black box. The concept is literally slide in, image out.
The Olympus FluoView® FV10i is essentially a confocal-in-a-box. Available in two configurations—the environmentally controlled FV10i-LIV for long-term, live-cell imaging and the FV10i-DOC for standard and oil-immersion work—the FV10i is a fully automated, four-laser confocal system intended for busy microscopy core facilities and space-conscious labs.
The FV10i, says Olympus product manager Brendan Brinkman, occupies less space than a standard confocal system; it can fit on any lab bench, even in well-lit laboratories. “So it helps bring the power of confocal microscopy to a wider audience, both because of its simplicity, its compactness, and because it doesn’t need a darkroom,” he says.
According to Brinkman, the FV10i features a simple icon-based navigation system that guides the user step by step through data acquisition in such experiments as 3D reconstruction and stem cell differentiation imaging. “Certainly when you’re talking about long-term time lapse, a dedicated instrument like the 10i-LIV is perfect,” says Brinkman.
That makes it a good choice for overworked microscopy core facility managers who might be spending more of their time training users to use high-end instruments for relatively routine projects and less time pushing those instruments to their fullest potential, he says.
“The FV10i was designed in part to offload that routine confocal work,” Brinkman says. “It’s a very high-end instrument, but it has a focus on just day-to-day kinds of routine experiments.”
Both instruments were upgraded in 2011. The FV10i-LIV costs $167,000, compared with about $180,000 for a standard Olympus confocal system, Brinkman says.
Initial resources
Not everyone favors the microscope-in-a-box concept. For one thing, for all its simplicity, what users lose is flexibility, such as the ability to add new laser lines or components. “The FV10i does 80% of what most people do on an instrument,” Brinkman says.
But perhaps just as importantly, researchers also lose the visceral experience of actually using a microscope.
“I am a microscopist,” says Olenych. “I value having a true microscope for the flexibility and field of view and the experience you get from looking through the eyepieces.”
For instance, he says, it can be harder to get a good overall feel for the sample when interacting with it solely on a computer monitor, as the field of view “is often many times smaller than what you can see in the eyepieces.” Also difficult to gauge is the sample’s “true” brightness or dimness.
That’s not to say microscopy isn’t difficult, Olenych agrees, but there are resources to help. As a first step, peruse Davidson’s Molecular Expressions website, which contains detailed tutorials on microscopy techniques and technologies, such as spinning disk confocals and super-resolution microscopy. “There’s a lot of great microscopy knowledge there. You can learn a great deal, and if that’s the first place you go, you are taking a good first step,” Olenych says.
Next, contact a company to discuss applications, expectations and needs and make sure you can find a system that provides the technologies you need today and the flexibility to grow in the future. “We don’t try to pigeonhole people into one technology,” Olenych says“We have a wide product basket that can meet their needs.”
Zeiss offers on-site training courses as well as periodic events like “Zeiss On Your Campus,” a traveling seminar series whose past topics have included live-cell imaging, general microscopy and materials science.
Olenych recommends that neophytes consider attending one of the many weeklong training courses at such locales as Cold Spring Harbor Laboratory and Woods Hole. “These courses are typically sponsored by the big vendors,” he says, “so it’s an easy place to see all the microscopes together and get a feel for how they work.” It also provides an opportunity to meet representatives from those companies and get a sense for your comfort level in working with them.
Simplicity through software
Of course users ultimately will have to, well, use a microscope. And no matter how you slice it, that can be complicated. “You cannot just sit down and snap a picture,” says Olenych. “The experiments are complicated, and you really have to have software to support the applications.”
Zeiss microscopes feature a software platform called ZEN (Zeiss Efficient Navigation), which was developed specifically to simplify common tasks, says Olenych. “People don’t like having to click five or six or 10 times to get an image,” he says.
1. So ZEN’s developers focused on getting users to results quickly, he says. For instance, with the “smart setup” tool, users enter the particular dyes they used on a sample, and the system configures itself to acquire those colors, moving optics in and out of the light path to collect, say, a DIC [differential interference contrast] image or perhaps a fluorescent one. Another tool, called “autoexpose,” adjusts laser power, detector gain settings and so on to quickly generate a test image and get the ball rolling.
Zeiss also includes a special maintenance objective that can be used, for instance, to test the instrument’s alignment. Like a printer-calibration tool, this objective tests the laser and scanner alignment and produces a quick green light/yellow light/red light indication of the instrument’s health. “A lot of people like that, especially core facility managers,” Olenych says. “It helps them determine if they need to call Zeiss, or if they can fix it themselves.”
Nikon Instruments Inc.’s NIS-Elements software platform is the control center for microscope system automation. According to William Jastromb, senior product manager for advanced bio-systems at Nikon Instruments, NIS-Elements is designed with an intuitive software interface that provides access to a broad range of tools for image capture, visualization and image analysis for both widefield and confocal microscopy. NIS-Elements is now shipping with Version 4.0, which adds several improvements and enhancements, with particular attention to ease-of-use options and advanced options for researchers with intricate protocols.
NIS-Elements, says Jastromb, is a popular addition to a core facility, because multiple users and projects with varying complexities can be managed, each user having a customized interface for his or her specific experimental configurations and devices. This allows all users to have a personalized view of their controls and settings, to repeat experiments with identical settings and to have all their tools and windows where they expect them to be.
“That makes it easier for people to get started,” says Jastromb, “and I think that’s where the activation energy, the major momentum is—how to get users started.”
Davidson is particularly fond of NIS-Elements’ interface design. “They put a lot of thought into how to make that software user-friendly,” he says. “It has many layers and can become extremely complex. But when you first start using it, the basic stuff is very easy.” Power users can drill down into the software for the fine hardware control needed for, say, super-resolution experiments. But simple acquisition experiments can be accomplished with just two or three button clicks, he says, and students can be taught to work independently in only 10 minutes.
In contrast, some third-party software, such as 3i’s SlideBook software, is “extremely kludgy,” Davidson says. SlideBook has all the power of NIS-Elements, but none of the intuitive simplicity, he says. “You have to drill down eight menus to get anything done.”
(Davidson, who has financial agreements with Nikon, Zeiss and Olympus to develop his microscopy websites, praises Zeiss’ ZEN software: “It’s still pretty new, but within two or three years I would say their software will rival Elements in ease of use.”)
Leica microscopes also are designed for use by both novice and expert users, says Leica marketing manager Chris Vega.
Leica’s DM (digital microscopy) concept is implemented in the DM (upright) and DMI (inverted) series of automated microscopes. These feature “intelligent automation,” Vega says, which enables the user to focus not on specific microscope components like filters, beam splitters and lasers but rather on the desired outcome.
“It’s really about making an easy-to-use interface for a user to get a desired result without being an expert microscopist,” Vega says. And the company’s microscopes, he adds, can help users achieve that goal even if the instruments are not enclosed in a box, “like the man behind the curtain in The Wizard of Oz.”
Leica’s DM microscopes, Vega says, were designed to work in imaging modes. “Instead of thinking of pieces-parts, users say, I want a DIC image, and you press the DIC button and the microscope configures itself so the customer doesn’t need to know [how to do that].” Another button press will reconfigure the instrument to take, say, a GFP [Green Fluorescent Protein] image, and the acquisition software then allows those two pictures to be overlaid.
Before you buy
Newbie users shouldn’t simply jump in and buy a microscope, Davidson advises. These instruments represent a significant investment—$150,000 and up, typically. “To get the funding for something like that, you have to have already made a splash in that area,” Davidson says.
In many cases, you can get your feet wet by working first with a collaborator or core facility. In Davidson’s case, his lab will perform pilot projects for users but requires more of a personnel investment (such as a graduate student) from the lab for longer-term projects.
If microscopy is going to become a more permanent aspect of your research program, and especially if multiple projects and lab personnel will be using it, it could be time to consider actually making a purchase. Davidson suggests systems like the Nikon C2, the Zeiss LSM 700 and the Olympus FV1000. “All are good entry-level confocals,” he says.
Or you can go even more general, with an instrument like the Zeiss Axio Observer, says Olenych. “You can configure that as a basic starting system that can do fluorescence imaging and transmitted-light imaging, and you can grow it all the way up to spinning disk or multiphoton or even a super-resolution system. So that stand has a lot of flexibility.”
The image at the top of this page is the FluoView™ FV10i Confocal Microscope from Olympus.