Getting to the Core

Hank Hogan, Contributing Editor

At Harvard Medical School in Boston, Jennifer Waters is quite popular at times, especially when she’s running a one-day training workshop for postdoctoral researchers. She and her staff teach the course every two weeks, and that isn’t often enough. “The demand is just unbelievable,” she said. “We have a constant wait list for all of the courses. We can’t teach enough of them.”

The insistent clamor isn’t motivated by valuable door prizes or an instructor’s theatrics. Instead, it’s driven by a desire to see clearly and image well on a small scale. Waters is the microscopy director of the Nikon Imaging Center at Harvard Medical School, and it’s her job to teach students how to use a microscope properly, take a digital image, align the illumination source correctly with the optics and do other necessary tasks.

Human epithelial cells were fluorescently labeled. The microtubules are shown in green, actin in blue and DNA in orange. The image was taken at the Nikon Imaging Center at Harvard Medical School. © J.C. Waters 2006.

Meeting the need for user education and training is a universal problem for core microscopy facilities. Technology presents another across-the-board challenge; specifically, acquiring it, maintaining it and keeping up with it. A third common issue is funding, which affects everything else. A look at sites across the country provides insight into what it takes to run a facility, and offers tips and guidelines on what to do and what to avoid.

Getting the picture in Boston

The Harvard microscopy center opened in 2001 and, as might be expected from the name, is full of microscopes, all on long-term loan from Nikon. The equipment includes spinning-disk confocals, total-internal-reflection fluorescence setups, standard confocal and upright microscopes, and an inverted fluorescence instrument. Along with the 11 microscopes, there are cameras, filters and heated chambers for live-cell work. Other corporate contributors of equipment include Hamamatsu Photonics, Diagnostic Instruments and PerkinElmer.

A postdoctoral fellow collects images with a spinning-disk confocal mounted on an inverted microscope at the Nikon Imaging Center at Harvard Medical School. Training in how to use equipment is often part of a core microscopy center’s mission. © J.C. Waters 2006.

Although Nikon provides a yearly grant in support of the center, Waters noted, the funding doesn’t cover all of her salary or that of her staff. Those costs are handled by the university, which encourages her to spend about 20 percent of her time on research. However, she isn’t required to seek grants for the research or to act as a principal investigator.

Experience has shown Waters that microscopy centers must consider two infrastructure issues: adequate ventilation, a fundamental requirement when many lasers are generating significant heat in a small space, and vibration isolation tables, which are vital for microscopes. “In most buildings, there is enough vibration to warrant them,” she said.

When it comes to finding people with expertise in overcoming such problems, Waters has had good luck with advice from fellow academics and vendors. She has found that the former have often faced similar challenges and that the latter can help with knowledge of a particular instrument or technique.

She also noted that core facilities are not ideal for new or inexperienced microscopy users in all research situations, citing, in particular, the needs for Förster resonance energy transfer (FRET) and calcium work. “Techniques that require a lot of analysis or preparation outside of collecting the images are difficult in a large core facility. Novices need one-on-one attention that is hard to provide.”

A farm with a different crop

Down the coast from Boston, the Howard Hughes Medical Institute (HHMI) has just finished building the Janelia Farm Research Campus in Ashburn, Va., near Washington. A first for HHMI, it will emphasize microscope development and neuroscience, as both relate to brain function and structure.

Doug Murphy manages the Janelia light microscope facility. “We’re starting out conservatively,” he said.

The facility will initially have six light microscopes along with associated setups for cell culture and histology, although there is room for much more equipment. Two of the microscopes will be confocal, and others will include a fluorescence-dissecting system and a motorized, inverted microscope capable of live-cell imaging.

The rooms housing the microscopes have been designed to minimize vibration and electromagnetic interference, either of which could adversely affect delicate instruments and experiments. Each room’s cooling system has been sized to handle the heat load of the lasers and illuminators it services.

Before joining Janelia Farm, Murphy ran the microscopy facility at Johns Hopkins Medical School in Baltimore. There, the facility recovered about 80 to 85 percent of its operating costs. Service contracts for the equipment alone ran to more than $100,000 per year. “You have to very carefully build out a plan for how to get those costs recovered, and one way that we approached it that seems to be working is to have a membership structure,” he said.

Memberships guarantee steady income to the facility while allowing dues-paying investigators access to microscopes at reduced rates. Murphy noted that there will be cost-accounting methods employed at Janelia Farm for microscopy and other shared resources. However, because HHMI is the sole research sponsor, the accounting will have different constraints and methods.

Finally, Murphy noted that, although microscope manufacturers have made progress in designing systems and software that are easier to operate, students and users who come to a facility need to know more than how to sit at a microscope and take pictures. He trains new users in a two-step approach: how to take a picture and how to get the additional information they require. “They need to actually know a little bit more about how the fluorochromes are operating, the acquisition parameters affecting a digital image and the fundamentals of image processing,” he said.

For his own ongoing training and to keep up with technology, Murphy depends on vendors, journals and trade magazines, and on the facility’s users. The first group provides information about and demonstrations of new equipment, but he finds users to be the best resource because they continually bring in new dyes, fluorescent proteins, imaging methods and even equipment when they use the facility.

In a library out West

On the other side of the continent at the University of California, Riverside, David Carter is the microscopy and imaging academic coordinator for a core facility found administratively, at least – within the botany and plant sciences department’s organizational structure. Physically, the equipment is on the bottom floor of what once was an abandoned library.

To keep users comfortable, the Leica lab at the University of California, Riverside, uses a negative-pressure ceiling vent to remove heat from its HeCd and visible argon lasers. The air flow prevents a laser failure when the blower on the argon laser seizes up. Hoses pass through the wall to a chiller unit to dump heat from the system’s UV argon laser. Courtesy of the Center for Plant Cell Biology, University of California, Riverside.

Building out and converting the library was interesting, Carter noted. There were a number of challenges in meeting all of the power requirements of the microscopes, and special care had to be taken to ensure proper disposal of the waste heat from the lasers. By installing adequate ventilation and relocating lasers when possible, things could be kept cool. This approach also reduced noise, since some of the lasers were moved out of work areas.

The facility houses five confocal microscopes and two stereomicroscopes. Carter noted that there are no vibration issues, unless there is an earthquake. The microscopes are from a mix of vendors, including Leica, Zeiss and Yokogawa.

There is a variety of other equipment, all of it in support of plant biologists, biomedical scientists, entomologists and a few users from other departments.

“The biggest users are plant cell biologists doing a lot of high-throughput imaging with GFP-tagged Arabidopsis seedlings,” he said.

Carter is a hands-on coordinator. For example, he designed a 48-well plate system for growing and imaging seedlings without having to touch them. He does some of the repairs himself, saving time and service-contract money. He prefers selecting instruments that won’t break easily. Nevertheless, service contracts are in place for systems deemed mission-critical.

And because of the long lead time for securing replacements, he buys spare lasers so that he can avoid the weeks of dead time that would otherwise result when a laser dies.

A point to remember, Carter said, is that vendors don’t test everything when installing a system. Thus, a core facility’s manager must run validation tests to make sure a piece of equipment does what it is supposed to do. However, even when tests are run, things get missed. “Minor problems can go unnoticed, and only pop up when a new user wants to use that feature,” he said.

That’s one reason why cultivating a good relationship with vendors is important, he said. Another motivating factor is that a core manager can’t be an expert on all of the equipment; vendors can help fill in knowledge and training gaps.

Up the coast

A day’s drive north of Riverside sits the Biological Imaging Facility at the University of California, Berkeley. It has a mixture of Zeiss microscopes, an Applied Precision deconvolution microscope and a Leica laser microdissection microscope. Director Steven E. Ruzin has been running the facility for 17 years. When it began, it was federally funded and essentially free for end users. The funding eventually ended, and the facility now charges a nominal fee for use.

This single optical section shows rat ovarian granulosa cells stained to visualize simultaneously the cytoskeletal proteins: F-actin (green) and alpha-tubulin (red) along with DNA (blue). Courtesy of Kenneth G. Geles, University of California, Berkeley.

Ruzin noted that fee-charging hasn’t turned the facility into a profit center. It still runs at a net loss. The fees, however, provide income that pays for service contracts, which he believes are essential. He also said that they caused users to take the equipment more seriously.

Newcomers must be trained by the core’s staff before they are allowed to use the equipment. The training varies according to the knowledge and experience of the user. Many only want to know how to take a picture and thus receive only that training.

Ruzin enjoys looking into a technological crystal ball. To do this successfully, he must first know his users, and then anticipate what they’re going to want two or more years down the road. That amount of lead time is needed to write grants, obtain funding, and buy and install equipment.

To successfully forecast future needs, he extracts information from vendors and scours the literature, looking for clues about where things are headed. It’s a fine balancing act, and sometimes that new equipment is delivered before it is in demand. “A couple of years ago, I bought a deconvolution microscope, not because we needed it at the time, but because we knew we would need it a year from then,” Ruzin said.

After the equipment arrives, it must be maintained. For the larger and more complex pieces of gear, those that can’t be fixed by Ruzin or by the staff, a service contract must be purchased.

In the end, service

In the center of the country is Chicago Medical School, which is part of Rosalind Franklin University of Medicine and Science. Daniel A. Peterson is manager of what might be termed an accidental core microscopy facility. More than seven years ago, Peterson, an associate professor of neuroscience, was a new investigator who wanted to make use of some rather expensive equipment. The only way to justify the cost was to spread it out over many potential users, and so a core facility was initiated.

Users of the core facility at Rosalind Franklin University have full access to confocal microscopy, bright-field microscopy and laser microdissection. Courtesy of Rosalind Franklin University of Medicine and Science.

His story isn’t that unusual. Many core facilities are created in this manner and eventually, if they survive, transition into something run by a staff.

Today the facility comprises two confocals, a laser microdissection system and a bright-field microscope. There are vibration isolation tables for microscope platforms, but the building is stable enough that there hasn’t ever been a need to use them, Peterson said. The facility has some 30 certified users at any given time. To be designated a user, individuals must undergo a training course. As in other facilities, certified users pay a subscription fee, an annual assessment per principal investigator.

Peterson noted that this cost-recovery structure covers most ongoing expenses, but that core facilities encounter other needs as they age, particularly after five or so years. At that point, computers and software must be upgraded, monitors burn out and other equipment fails. The money for such repairs and upgrades comes from the university, which also supports a portion of staff salaries.

Additional costs arise as new technology emerges. For example, spinning-disk confocal imaging was not up to speed when the core facility was originally set up, he explained. That situation has changed. “Several microscope manufacturers have since improved the utility of the spinning-disk confocal imaging, and we would be interested in obtaining one of them, but now we have to find another source of funding,” he said.

Before starting a core facility, Peterson noted, it is important to consider certain questions, such as, who will provide the necessary long-term commitment? Will the core’s creator and likely manager provide technical support or act as an expert consultant? Will the demands prove too draining, especially for someone who is also a principal investigator with research to do and grants to obtain?

Despite the burdens, however, Peterson said that running a core microscopy facility can be a truly rewarding experience; for instance, when a graduate student makes significant progress on a project because a little guidance and expertise helped overcome a problem that had been holding the research back.

“You have to live for those moments of reward to offset the responsibility of trying to keep a good core facility running,” he said.