It all starts with the breach of a single cell. Viruses, bacteria, and other maladies-in-waiting skulk around the body, waiting for a chance to strike. Once they slip past the immune system’s defenses, they start ferociously multiplying, preparing to unleash havoc on an unsuspecting host.
Exactly how and why the cell’s defenses fail against some invaders while successfully fending off others has long been a mystery. A novel super-resolution microscopy technique developed at Sandia is providing new answers to old questions by unveiling never-before-seen detail of the cell membrane. The insights gleaned from the research could open doors to new diagnostics, prevention, and treatment techniques. The research supports Sandia’s biological threat reduction programs and could be expanded to support biofuels research.
“We’re trying to do molecular biology with a microscope, but to do that, we must be able to look at things on a molecular scale,” says Jesse Aaron (8622), a postdoctoral appointee.
The cell membrane is a bustling hub of activity on a miniscule scale. While providing structure and housing the cell’s interior, the membrane regulates movement of materials in and out of the cell, controls adhesion to other objects, and coordinates the cell’s communications and subsequent actions through signaling. Receptor proteins on the surface of immune cells, known as Toll-like receptors (TLRs), are tasked with recognizing intruders, or antigens. The TLR4 member of this receptor family responds to certain types of bacteria by detecting lipopolysaccharides (LPS) present on their surface. They subsequently initiate signaling to alert the cell and activate an immune response.
A vexing problem
Using imaging techniques they developed, Jesse, Jeri Timlin (8622), and Bryan Carson (8622) discovered that TLR4 proteins cluster in the membrane when confronted with LPS derived from E.coli, which increases cell signaling and response. Interestingly, LPS derived from the bacteria that causes plague, Yersinia pestis, do not cause the same effects. This finding, which marked the first time such small events could be imaged and compared, could explain why some pathogens are able to thwart the human immune system.
Being able to image the cell surface with high enough resolution to see the earliest binding events has been a vexing problem, since even the most sophisticated optical microscopes are bound by the diffraction barrier, which limits what can be resolved using visible light.
“With more traditional visualization methods, you can’t see the level of detail you need. It’s important to look at not only what’s present, but also when and where it’s present in the cell,” says Jeri.
Dual color capabilities
The technique used by Jeri and Jesse builds on superresolution capabilities developed in recent years, but goes another step by adding dual-color capabilities to the relatively new stochastic optical reconstruction microscopy, or STORM. The combination enables the Sandia team to get an exponentially better picture by simultaneously imaging LPS and TLR4 receptors on the membrane.
“Current capabilities are akin to looking out the window of an airplane and seeing the irrigation circles. You know that plants are there, but you can’t tell what kinds of plants they are or what shape the leaves are,” says Bryan, a Sandia immunologist who was an integral part of the project. “But with this technology, it’s like zooming in and seeing the leaves and the structure of the plants. That buys you a lot in terms of understanding the mechanism.”
The NIH awarded Jeri a five-year, $300,000 per year grant in 2009 to develop such visualization power, and it has exciting potential for future applications. Next on the agenda is developing the capability to image live cells in real time using spectral stimulated emission depletion, or STED, technology.
“We’re working toward using a version of superresolution that’s much more live-cell friendly, and extend that in terms of what colors are available to do multiple colors, but still maintain the live-cell friendliness. I see this as a beginning of a long development in this type of imaging technology,” Jeri says.
The eventual goals will likely expand as the technology reveals additional surprising capabilities. Eventually, the Sandia team would like to be able to visualize protein/protein interactions.
Seeing the whole biological process
“Every biological process that goes on in your body is somehow controlled by proteins forming complexes with other proteins or complexes in the membrane, so this would give you this ability to look, with high spatial resolution and multiplexed color capabilities, at four or more things in a living cell, which can’t be done very easily right now. It can be done in pieces, but we want to see the whole biological process,” Jeri says.
The technology has exciting potential in immunology and drug discovery as well, Bryan says.
“We’re hoping to do something like label the viral particles and watch them in real time, or as close as we can to real time, in the internalization process,” he says. “With the super-resolution technique, we can actually watch them move through the membrane and see if there are other structures being recruited by the virus to the site of internalization. That will hopefully give us mechanistic insight into how a given virus enters a cell. Understanding that mechanism can lend itself to identifying drugs or other compounds that might block viral entry.”
The team is interested in expanding the technology’s capabilities to research other areas such as biofuels to better understand where and when different pigments are located on the membrane of oil-producing algae. This would provide valuable insight into their photosynthesis functions, which could help in more efficient biofuel production.
“A lot of this work is still pretty initial, but we’re encouraged by what we’re seeing and excited for future potential,” Jesse says. - Stephanie Hobby
By Patti Koning
What if a nuclear reactor melted down, exposing the nearby population to dangerous levels of radiation? That scenario has been on everyone’s mind since Japan’s devastating 9.0 earthquake and subsequent tsunami crippled the Fukushima Daiichi nuclear plant.
“The trouble with radiation sickness is that the symptoms are often latent and don’t appear for days, weeks, or even months,” says Greg Sommer (8621). “But data clearly shows that if you can start treatment within the first 24 hours of exposure, the prognosis for recovery goes up significantly.”
Radiation exposure leaves a clear signature in a victim’s blood, but reading that signature is time- and labor-intensive, far outside the scope of an emergency response. For the past year and a half, Greg has been working on a Laboratory Directed Research and Development (LDRD) project to create a handheld radiation biodosimetry device to answer that need.
“In a mass exposure, you can’t rely on hospitals and clinics for screening because demand will quickly overwhelm their capacity,” he says. “The goal is to put a drop of blood into the device without any pretreatment — it could come straight from someone’s finger — and have an answer about their level of exposure within 15 minutes. Then you can target therapeutics to the people who really need them.”
The original LDRD project set out to develop a portable device to rapidly screen blood for a panel of protein biomarkers, leveraging Sandia’s expertise in point-of-care clinical diagnostics and protein screening. That scope changed after Greg began working with colleagues at the Armed Forces Radiobiology Research Institute (AFRRI), a DoD laboratory, who have developed the panel through 30 years of research on radiation signatures in blood using animal models.
“They asked us to bring in a hematology component to do a white blood cell count,” he says. “A good white blood cell count combined with the protein measurement yields a very accurate dose assessment. This threw a monkey wrench into our project, but it’s a worthy challenge. Point-of-care hematology is a powerful tool.”
Meeting two daunting challenges — adapting hematology to a portable format and then combining methods for protein detection and white blood cell counting onto a single device — required a whole new approach, something quite different from the standard microfluidics-based lab-on-a-chip used in many of Sandia’s portable diagnostic tools. The answer to both challenges, it turns out, was centrifugal force.
Centrifugal force is key
Postdoc Ulrich Schaff (8621), who did his doctoral research at a University of California, Davis lab studying inflammation and white blood cells, turned to the concept of lab-on-a-disk, which uses the centrifugal force generated by a spinning disk to manipulate a sample.
“The idea was to put a preparatory centrifuge in the device to do blood counts directly from the separation technique,” Ulrich says. “We’ve combined operations that you really can only do on a disk. If you just try to miniaturize benchtop preparation methods, you wind up with a very complex network of channels and storage containers. Using separation principles for these steps greatly simplifies the design.”
The lab-on-a-disk prototype that Greg and Ulrich developed is deceptively simple, in that it hides their meticulous work developing disk-based assays and the mechanics of the disk itself. First the sample, a drop of blood, is routed to the center of the disk, which is then spun slowly to enable capillary action to route the sample through channels into different assay regions. The sample is mixed with pellets to capture the proteins and fluorescent tags that enable identification. A faster spin of the disk sends the different samples to the bottom of the channels for analysis.
The beauty of the device, says Greg, is that it is simple and ubiquitous.
“All you need to run the disk is a motor and a laser, both found in any CD player,” he says.
Designed to run on existing infrastructure
That simplicity was quite clear as they developed the disk using a Dremel tool as a motor and a hairdryer to simulate a heating element. They read the results under a microscope, a step that will be automated using a laser to produce a digital readout.
Ulrich and Greg published initial results in Clinical Chemistry (Vol 57, Issue 5, pp 753-761, 2011) demonstrating protein separation using the lab-on-a-disk. A follow-up publication is planned to show that the device can also perform white blood cell counts.
They now are using DoD blood samples to validate the device’s analytics against “gold standard” benchtop methods. The team hopes that getting the lab-on-a-disk radiation biodosimeter into the field won’t be difficult, given its overall simplicity.
“The system is being designed to run on existing infrastructure, so there wouldn’t be the typical barriers of capital investment or training,” says Greg. “With the hematology component, this device also has potential for applications far beyond radiation biodosimetry, such as biodefense, emerging infectious diseases, cancer, and HIV treatment and research.” -- Patti Koning
Sandia’s Battery Abuse Testing Laboratory is a center of mayhem and destruction on a good day, and it’s about to get even better. The nation’s go-to center for battery testing was built in 1991, and since then has conducted critical scientific studies to evaluate the safety of thousands of batteries, including 12 years of testing for the FreedomCAR program and the US Advanced Battery Consortium.
The one-of-a-kind facility analyzes performance under any number of abuse scenarios batteries might face in the real world, and it’s getting a $4.2 million renovation. The overhaul will further the lab’s capabilities as part of a national stimulus package to develop low-cost batteries for electric and plug-in hybrid electric vehicles. The funds are being used to update test bays, data acquisition systems, and laboratory space, and additional staff members have been hired to meet the growing demand for Sandia’s battery safety expertise.
“This will bring our capabilities up to the point where we can test larger batteries that are going to be relevant to the electric vehicle market, and move up to batteries that will be used in plug-in hybrid electric vehicles,” says Chris Orendorff (2546), team lead for the Battery Abuse Testing Lab. “We’ll have the capability to test batteries in the 5- to 15-kilowatt-hour range, which we’ve never done before. This scale of testing is critical to the deployment of electric vehicles that are needed to reduce the nation’s dependence on foreign oil.”
Developing capabilities in clean energy
During a visit to Sandia in November 2009, Deputy Secretary of Energy Daniel Poneman announced the Battery Abuse Testing Lab funding as part of a $104.7 million stimulus package. The goal of the package is to further develop the nation’s efforts in clean energy and efficient technologies across seven DOE national labs. Sandia’s portion is paying for much-needed upgrades while supporting several new lab positions and sustaining about 50 construction, architectural, and engineering jobs.
"This has been a great way to do our part in putting people in the community to work and keep them working," says Charles Tomlin (4827), construction manager for the project. "We've worked with 11 architects and engineers and about 30 to 40 construction contractors and vendors, and we expect to be done with construction three months ahead of schedule."
The upgrades include an X-ray computerized tomography system that will generate 3-D images to allow researchers to conduct failure analysis without doing physical analysis, which can be destructive. The lab’s battery calorimetry capabilities will be the world’s largest and will include six accelerating rate calorimeters (ARCs), three isothermal battery calorimeters, one microcalorimeter, and one differential scanning calorimeter, all of which will be consolidated and housed in the new facility. New spectrometers and laser diagnostics for gas measurements, upgrades to the scrubber system, and additional battery cyclers, supporting higher-energy batteries, are also on the lab’s roster of new equipment.
“Chris and his team are already internationally recognized for their work. The recapitalization will allow us to sustain that leadership position in battery safety research and continue to develop new diagnostic techniques that are needed by domestic automotive manufacturers and their battery suppliers,” says Tom Wunsch (2546), manager of Sandia’s battery research efforts.
Need for upgrades readily apparent
Being the nation’s leading battery abuse testing center for the past two decades has taken its toll. Inside the 2,000-pound blast doors, the need for upgrades is readily apparent. The test bays bear witness to the years of battery abuse testing, which can result in smoke, fires, and violent decomposition events. Much of the equipment is original and needs to be modernized and upgraded to meet the nation’s growing energy storage needs.
The remodeled bays are completely stripped clean, coated in an epoxy paint to make clean-up easier, with new explosion-proof lights and a new CO2 fire suppression system that can be manually or automatically engaged to quickly bring any large fires under control.
“In addition to the fire suppression system, we have moved all of the live power out of the test bays, except for the temporary power required for any given test. This allows us to safely cut power to the unit [being tested] should safety concerns warrant,” says Bill Averill (2546), who oversees day-to-day operations of the lab while providing technical battery testing support.
New data acquisition systems will ensure a much more precise readout of results. The new systems will also help with efficiency, reducing set-up time by as much as a day, which lab leaders say will increase throughput by a factor of six. “The bays will be hard-wired and ready to go, so we can bring in batteries, connect them to the testers, and start testing,” says Chris. “We can also run two tests simultaneously, which we’ve never been able to do before. These kinds of streamlined test capabilities will help expand our customer base, increase throughput for the lab, and will enable us to provide more support for industry.”
Because much of the battery lab’s testing is done for external clients, the area outside the control room will have two new 42-inch monitors so visitors can watch the test from outside the control area.
American jobs, American equipment
Although the lab is unmistakably a construction zone, testing is still being conducted in half the lab while the other half is overhauled. Construction crews are there from early morning until the early afternoon, at which time the laboratory team sets up and conducts tests.
Construction started during the 2010 winter shutdown, with completion scheduled for September 2012, but the work will likely be complete in June 2011, and Chris anticipates that the lab will be fully operational by March 2012.
“Because these are Recovery Act funds, we realize the importance of trying to get this spent on American jobs and American equipment. We are doing everything we can to get that done as quickly and responsibly as possible,” Chris says. About half of the equipment funds were spent within six months of beginning the project. “Getting this money out into the economy is one of the DOE’s priorities, and we’ve worked pretty hard to do that.” - Stephanie Hobby