Almost There:
Cutting-Edge Molecular Microscopy Center Prepares to Open

By Jason Socrates Bardi


"It is very easy to answer many of these fundamental biological questions; you just look at the thing!... Make the microscope one hundred times more powerful, and many problems of biology would be made very much easier. I exaggerate, of course, but the biologists would surely be very thankful to you—and they would prefer that to the criticism that they should use more mathematics."

——Richard P. Feynman. From There's Plenty of Room at the Bottom, a lecture given to the American Physical Society in 1959.


When Associate Professors Bridget Carragher and Clint Potter arrived at The Scripps Research Institute (TSRI) last year, they knew where their laboratory space would eventually be, but they had no idea what the space would be like. That is, until after they sat down with Professor Ron Milligan over drinks one night and drew up plans on a blank blueprint of the interior of the CarrAmerica B building.

In the following weeks, this became the blueprint for Milligan's dream of the most advanced biological microscopy center in the world—the Center for Integrative Molecular Biosciences (CIMBio)—which officially opens next month. CIMBio is built around its advanced microscopes and open laboratories and houses several TSRI faculty under one roof.

"We had an almost unique opportunity to design the best electron microscopy suite, and we put a lot of effort into doing this," says Milligan.

The design is predicated on six rooms for microscopes, which are at the center of the building. The microscopes are mounted on three-foot-thick concrete slabs isolated from the building's foundation, which protect the instrumentation from vibrations. The rooms are climate-controlled with low humidity to prevent contamination of samples by water vapor, and they are sound-proofed so that noise from the corridors does not cause vibrations. The air supply coming into the rooms passes through a nylon sleeve that breaks up any air currents, and the microscopes can be controlled entirely from a separate room so that the samples can be left alone in the dark under the microscopes.

"It's quiet, there are no air currents, and the microscopes are sitting on a very stable platform," says Milligan.

Ground broke on the interior design of CarrAmerica B in March, and the construction lasted throughout the fall. The first groups moved in at the end of December. A year ago, CarrAmerica B was a shell. Now it is an oyster with more than one pearl.

Molecular Machine Mania

Milligan, Carragher, and Potter are all founding members of the CIMBio, which was organized to combine the talents of several groups across campus.

The center seeks to speedily obtain and analyze high-resolution structural images of large molecular complexes of the cell by combining the use of x-ray crystallography and electron microscopy (EM). CIMBio members include investigators Francisco Asturias, M.G. Finn, Jack Johnson, Elizabeth Wilson-Kubalek, Mari Manchester, Nigel Unwin, and Mark Yeager.

What unites the members of CIMBio is their interest in the combined use of the x-ray crystallography and EM techniques as a means to unravel the structure and mechanism of action of the large molecular assemblies of the cell—such as the transcription complexes that make messages from the genes, membrane channels and pumps that import and export materials, and the tiny molecular tracks and motors that move cells and form important structures like the mitotic spindle.

Phase I of CIMBio will be devoted to working out the structure of the proteins and nucleic acids in complexes that carry out the work of the cell.

While the individual protein components of these machines may be studied by x-ray crystallography, the machines themselves are compositionally and conformationally dynamic, making them unsuitable for x-ray methods. They are, however, ideal specimens for electron microscopy. Polymerases, membrane complexes, viruses, and motor proteins can all be visualized in their native environment using EM.

Phase II will concentrate on the dynamics of those cellular machines—their assembly, disassembly, and control over time.

Laboratory space for that effort is already under construction in CarrAmerica B, and at the end of the year, investigators Velia Fowler, Klaus Hahn, Clare Waterman-Storer and Kevin Sullivan will relocate there to lead the Phase II efforts.

The building combines several of these laboratories into one contiguous shared space built above and around the microscopes. The laboratories have an open design and some of the facilities—like the microscopes and an imaging area—are shared, something that the CIMBio researchers appreciate.

"This is a collection of widely diverse scientists, and we want to maintain and enrich our collaborations" says investigator M.G. Finn, whose group was the first to move into the new space. "Here we can't help running into each other."

EM Imaging of Biological Structures

Electron microscopy, which has been around since the 1930s, uses a beam of electrons to image tiny objects onto a digital camera or a photographic plate. EM looks at a range of magnifications, from no more than an ordinary microscope that magnifies up to 60 times to those that magnify up to 1,000,000 times. CryoEM, which is the technique used for viewing biological materials, requires the samples to be spread to a thin film and frozen on a copper meshwork grid.

The final products of these electron images are 3-D maps, which are representations of the cellular structures on the slide at near-atomic resolutions—up to about 3-4 angstroms under the best of circumstances. When combined with the x-ray structures of the component parts of the structures, EM maps can yield a detailed description of the structure and action of the entire machine.

Further application of this technique will be an invaluable tool for studying membrane-bound proteins, which are notoriously hard to crystallize. Less than one half of one percent of the structures contained in the Brookhaven National Laboratory Protein Data Bank are of integral membrane proteins, despite the fact that over a third of all proteins in the body are in the membrane.

But EM is not a routine technique. Calculating an EM structure manually takes weeks or even months. It can be tedious.

A single high-resolution image of a sample under an electron microscope has too much noise to yield accurate molecular representation. Images must be averaged together with their counterparts to reduce noise. Plus, any single molecular assembly imaged will be but one 2-D projection of what is a 3-D object, so the averaging must be done over many possible angles. To build a 3-D model, one must take many images and build a structure by looking at all the different angles of all the different molecular assemblies imaged.

Building a 3-D model is like looking at a piece of sculpture in a gallery. Only by walking around the piece and viewing its various sides and angles can the brain build a mental image of the art and fully comprehend its dimension, perspective, and scale. The same is true using a computer. Only by piecing together many different views of a molecule from a microscope can a computer build a model of the molecular assembly.

And the molecule that is being imaged gets destroyed in the process, so the next image must be captured from some other part of the sample holder grid. This has always required a person to choose different spots on the grid manually. As the number of grid spots goes up, so goes the level of tedium.

"What we really want is 100,000 to 1,000,000 molecule images and that just takes too long to do manually," says Carragher. "Then you want to do 10 different conformational states, 20 different labeling studies, and each time it's going to take three to six months. That's more than the lifetime of a graduate student."

"There are projects," Carragher adds, "projects people just don't do because the manual labor required is just too daunting."

Carragher and Potter, who lead the Automated Molecular Imaging group, are creating algorithms for automated data collection and analysis, which should simplify the technique of electron microscopy and enable throughput to be increased dramatically.

So Long, John Henry

Several years ago, Carragher and Potter suggested that automated data collection and analysis could be developed for EM. A similar goal had been accomplished in x-ray crystallography, and given the need for structural information in our post-genomics proteomics world, automation would represent significant progress.

So Carragher and Potter started developing automated EM algorithms and began writing grants with Milligan to develop these into programs. "It took off from there," says Carragher.

They succeeded in developing software for both the collection and the analysis, which they brought to TSRI when they came last year to form the Automated Molecular Imaging group at TSRI. Milligan helped recruit his long-time collaborators from the University of Illinois at Urbana–Champaign, where they were co-directors of the Imaging Technology Group of the Beckman Institute for Advanced Science and Technology.

Creating the algorithms was not easy. Using the manual technique, a person has to make decisions about where to focus the EM beam and take a picture, looking first at low resolution and then deciding in which areas to collect data at high resolution. For automation to succeed, the computer must do the same thing and use intelligent criteria to search the low resolution image for appropriate targets.

"Even a two-year-old can tell a cat from a dog, but that's a very hard problem for a machine," says Carragher. "But what humans are not good at is doing the same boring thing a thousand times in the dark for weeks."

Carragher and Potter had to write their software to take a low-resolution image, select areas to image in medium resolution, and then analyze that image and strip out targets for high-resolution maps. Then, they had the computers put the data into processing programs and calculate 3-D maps. Recently, they have been testing and refining the programs.

"What we have done over the past year is to show that you can insert a [sample] in the microscope and [calculate] a 3-D map fully automatically," says Potter.

In fact, Carragher and Potter constructed one of the best 3-D maps of the tobacco mosaic virus in under two days. By comparison, the work would have taken several months of work just a few years ago and perhaps several weeks using conventional methods today.

"We can now go from inserting a specimen in the microscope to having a 3-D map in 24 hours," says Milligan, adding that the fear of failure should no longer be a limiting factor for experiments.

Still, the automation is not fully implemented, so one of the immediate goals of the Automated Molecular Imaging Group is to see their software used for practical applications, something that their coming to TSRI will facilitate.

"There are so many people who want to collaborate with us here—it's great," says Potter, adding that within a few months of their arrival they had already found an almost overwhelming number of projects.

"At the moment we need to make the technique very efficient and very general, [and] get it out to the community" says Carragher. "We can do it, and now we want to be able to do it routinely for anybody."

Additional plans include the design of technology that would make EM high-throughput. This includes a robotic specimen handler that Carragher and Potter have been experimenting with that would allow the instruments to be left alone to collect and analyze even larger sets of data.

"You could look at maybe 10 grids overnight," says Potter.

Ready for Tours

Though the shared space of the CarrAmerica B building and the collaborations it fosters within CIMBio and throughout the TSRI campus will be reward enough, there is one more thing that the building provides: ready-made tours.

In the plans that Milligan and the others drew up, they envisioned several people controlling the microscopes and discussing the images as they are collecting data. They also anticipated people peering through the glass wall of the control room, and this has its advantages.

"It's a very easy way to communicate what we are doing," Carragher says to me as we walk past the control room on a recent tour of the facilities. A technician had one of the new microscope's back panels open and was busy fiddling with some wires.

"Is it done yet?" Carragher asks.


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Professor Ron Milligan had a dream of building the most advanced biological microscopy center in the world. Milligan, standing, is seen here working with MCSC graduate James D. Jontes. Photo by Michael Balderas.






Associate Professor Bridget Carragher (above) is working with Associate Professor Clint Potter to create algorithms for automated data collection and analysis, which should simplify the technique of electron microscopy and enable throughput to be increased dramatically. Photo by Kevin Fung.






"There are so many people who want to collaborate with us here—it's great," says Potter. Photo by Quan Dong.








This model of tobacco mosaic virus at 10 angstrom resolution was generated completely automatically from specimen grid to 3-D map in less than two days.







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