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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