Synchrotron Infrared Structural Biology Program

http://www.src.wisc.edu/news/stories/Tomography.08.12.13.htm

Transforming Science at SRC with Nondestructive Synchrotron FTIR Spectro-Microtomography

August 12, 2013
Chris Moore, cmoore@src.wisc.edu
Researchers at the Synchrotron Radiation Center (SRC) have developed, for the first time, Fourier transform infrared spectro-microtomography.  This powerful technology allows for nondestructive three-dimensional imaging that reveals the distribution of distinctive chemistry throughout an intact biological or materials sample.  By yielding spectral data for each voxel of the 3D sample, it is now possible to easily characterize, for example, fragile cell structures throughout a cell body.  This work is reported in the Journal Nature Methods, and was the result of a collaboration between SRC Users Carol Hirschmugl from UW-Milwaukee and Michael Martin from Lawrence Berkeley National Laboratory.

Figure 1: Corresponding authors Carol Hirschmugl and Michael Martin at an SRC Users’ meeting.  (Photo by  Chris Moore, SRC)
The development of this new technique was the result of a convergence of expertise and technology at SRC including rapid, high-quality, wide-field 2D infrared imaging at the microscale. The synchrotron light is critical, providing high signal to noise. The key to this technique is the existence of a unique instrument called the Infrared Environmental Imaging (IRENI) beamline at SRC.  Hirschmugl and SRC staff developed this high spatial-resolution infrared imaging instrument with funding from the National Science Foundation. 
Hirschmugl says, “The critical components of IRENI for IR tomography experiments are that it collects 12 beams of synchrotron infrared light to homogeneously illuminate a 128 x 128 array detector (like a CCD camera for infrared) for which high quality spectral data is rapidly collected for every pixel in parallel.”  Spectral data, which are variations of the absorption of IR light for every wavelength, are used to determine the molecular structure of a sample.
Hirschmugl and Martin applied existing computed tomography reconstruction methods (CT-scans) to frequency dependent data from the IRENI instrument yielding spectral information for every voxel.  A voxel is a 3D volume element, analogous to a pixel in 2D.  When carefully rotating a sample, 2D projections of spectrally rich data at many angles were collected. Millions of voxels of data containing  spectra data were extracted using CT reconstruction algorithms that were modified to evaluate all wavelengths, and this powerful new technology was born.

Figure 2: Test sample rotation stage at the SRC IRENI beamline used to take 3D IR Tomography data. (Photo by C. Hirschmugl, UW-M)
The significance of this new technology cannot be overstated.  “Existing methods of studying the delicate structures within a cell include slicing, staining, chemically labeling, and adding contrast agents, techniques that modify cell structures in some way,” says Hirschmugl.  With this technique scientists can now study essential cell functions within living tissue including their architecture, mechanical movement, biochemical triggers, and cell-to-cell communication, all without affecting the living system under study. 
In their journal publication, Hirschmugl and Martin showcased the measurement, 3D reconstruction, and composition of a single Zinnia elegans cell, a sliver from a fast-growing Populus tree, the internal structure and composition down the axis of a human hair, and the biochemical structure of a colony of mouse stem cells.  A better understanding of the structure and composition of cells like the Zinnia or the Populustree is needed for making biofuels from cellulose.  Analysis of a human hair revealed a distinctive biochemical construction, including different distributions of material down the axis of the hair, while imaging the stem cell colony showed the ability of this technique to analyze in 3D crucial delicate structures impossible to study in any other way.

Figure 3: A sliver of populous wood (a), two 3-D reconstructions (b, c), and 3 cross sections (d– i).  The analysis and reconstruction, made possible by co-authors Barbara Illman and Julia Sedlmair from the Forest Products Laboratory, was done without destroying (cutting) the sample.
Future work by Hirschmugl and Martin include plans to improve the efficiency of data collection and analysis and inclusion of advances in other fields to automate the collection, processing, and storage of large spectra tomographic data sets.  Additional improvements will also result from continued advances in mid-IR array detector technologies and synchrotron IR beamlines.  This new powerful method will facilitate a wide variety of scientific, industrial, materials, energy and medical applications ranging from understanding how to produce biofuels to the functioning of stem cells.
Co-authors participating in this experiment provided expertise in tomographic reconstructions and samples of interest and sample preparation.  They include colleagues from Forest Products Laboratory (Barbara Illman, Julia Sedlmair), UW-Milwaukee (Miriam Unger), Lawrence Berkeley National Laboratory (Charlotte Dabat-Blondeau, Hans Bechtel, Dilworth Parkinson), University of Mainz (Jonathan Castro), Lawrence Livermore National Laboratory (Marco Keiluweit), University of UW-Madison (David Buschke, Brenda Ogle), and Karlsruhe Institute of Technology (Michael Nasse). 
This research was funded by the DOE Office of Science and the National Science Foundation.  Berkeley Lab’s ALS and NERSC are funded by the DOE Office of Science.  SRC is primarily funded by the University of Wisconsin-Madison, with supplemental support from facility users and the University of Wisconsin-Milwaukee. 
Additional Information:
News release from Berkeley Lab
http://newscenter.lbl.gov/news-releases/2013/08/05/3d-ir-images-now-in-full-color/
Nature Methods Paper (a subscription may be required)http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2596.html************
The Synchrotron Radiation Center (SRC) of the University of Wisconsin-Madison is a national light source facility providing infrared, ultra violet, and soft X-ray light for use in research on exotic materials, ranging from high temperature superconductors and computer chips to cancer cells.  SRC provides an environment uniquely suited to the performance of seminal research, the development of new experimental techniques and instrumentation, and the training of scientists for the future.

3D IR Images Now in Full Color

Berkeley Lab and University of Wisconsin Researchers Unveil FTIR spectro-microtomography

Researchers at Berkeley Lab and the University of Wisconsin-Milwaukee have reported the first full color infrared tomography. (Image by Cait Youngquist)

http://newscenter.lbl.gov/news-releases/2013/08/05/3d-ir-images-now-in-full-color/

Lynn Yarris (510) 486-5375

An iconic moment in the history of Hollywood movie magic was born in the 1939 film The Wizard of Oz when Judy Garland as Dorothy Gale stepped out of the black and white world of Kansas into the rainbow colored world of Oz. An iconic moment in the history of infrared imaging may have been born with the announcement of the first technique to offer full color IR tomography.
A collaboration between researchers at the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of Wisconsin-Milwaukee (UWM) has combined Fourier Transform Infrared (FTIR) spectroscopy with computed tomography (CT-scans) to create a non-destructive 3D imaging technique that provides molecular-level chemical information of unprecedented detail on biological and other specimens with no need to stain or alter the specimen.
“The notion of having the colors in a 3D reconstructed image being tied to real chemistry is powerful,” says Michael Martin, an infrared imaging expert at Berkeley Lab’s Advanced Light Source, a DOE national user facility. “We’ve all seen pretty 3D renderings of medical scans with colors, for example bone-colored bones, but that’s simply an artistic choice. Now we can spectrally identify the specific types of minerals within a piece of bone and assign a color to each type within the 3D reconstructed image.”

Michael Martin at Berkeley Lab’s Advanced Light Source (Photo by Roy Kaltschmidt, Berkeley Lab)

Martin is one of two corresponding authors of a paper describing this research in the journal Nature Methods titled “3D Spectral Imaging with Synchrotron Fourier Transform Infrared Spectro-microtomography.” The other corresponding author is UWM physicist Carol Hirschmugl, Director of the Laboratory for Dynamics and Structure at Surfaces and a principal investigator with UW-Madison’s Synchrotron Radiation Center (SRC).
Every individual type of molecule absorbs infrared (IR) light at specific wavelengths that are as characteristic as a human fingerprint. IR spectroscopy can be used to identify the chemical constituents of a sample and the application of the Fourier-transform algorithm allows all IR fingerprints to be simultaneously recorded. FTIR spectroscopy is especially valuable for imaging proteins and other biological samples because it is non-destructive and can be performed without altering the sample. Martin and Hirschmugl and their colleagues have combined FTIR with computed tomography, the technique for reconstructing 3D images out of multiple cross-sectional slices, to achieve what is believed to be the first demonstration of FTIR spectro-microtomography.

Carol Hirschmugl at University of Wisconsin’s Synchrotron Radiation Center

“FTIR spectro-microtomography involves low-energy IR photons that do not affect living systems and do not require artificial labels, contrast agents or sectioning,” Hirschmugl says. “It greatly enhances the capabilities of both FTIR spectroscopy and CT by creating a full-color spectro-microtomogram in which each voxel contains a complete spectrum (millions of spectra per sample) that provides a wealth of information for advanced spectral segregation techniques such as clustering, neural networks and principal-component analysis.”
The success of FTIR spectro-microtomography was enabled by the speed with which 2D FTIR images can be obtained at the SRC’s Infrared Environmental Imaging (IRENI) beamline. The SRC is a synchrotron radiation facility that provides infrared, ultra violet, and soft X-ray light for scientific research. IRENI offers one the nation’s highest performance IR imaging beamlines through the use of unique focal plane array detectors.
“With capabilities such as those at IRENI, we can obtain hundreds of 2D spectral images as a sample is rotated,” Martin says. “For each wavelength, we can then reconstruct a full 3D representation of the sample via computed tomography algorithms.”
Martin, Hirschmugl and their colleagues developed a motorized sample mount that precisely rotates the sample while holding it at the focus of an IR microscope. Data col­lection of 2D spectral transmission images as a function of sample angle is automated, and the computed tomography algorithms allow full reconstructions for every wavelength measured that are then reassembled into a complete spectrum for every voxel.
“We’ve been able to do a lot of exciting science with 2D FTIR imaging at the diffraction limit using synchrotron infrared beamlines, and it’s very exciting to now be able to expand this to true 3D spectroscopic imaging,” Martin says. “While the most immediate applications will be in biomedical imaging, I think full color FTIR spectro-microtomography will also be applicable to imaging 3D structures in biofuels, plants, rocks, algae, soils, agriculture and possibly even studies of art history where different layers of paints could be revealed.”

Spectro-microtomographic images of a human hair show absorptions of protein (red) and phospholipid (blue-green). Center, the medulla is observed to have little protein. Bottom, the medulla has higher concentrations of phospholipids.

The Berkeley Lab and UWM researchers have already successfully applied FTIR spectro-microtomography to obtain 3D images of the molecular architecture of the cell walls in a flowering plant – zinnia – and in a woody plant – poplar. A better understanding of the chemical composition and architecture of plant cell walls is critical to the ultimate success of making biofuels from plant biomass. The collaboration also applied FTIR spectro-microtomography to study human hair, which has a distinctive biochemical construction, and an intact grouping of pluripotent mouse stem cells.
“The hair study showed that spectral reconstructions can be done on larger fully hydrated biological samples and that we can spectrally identify a fully buried portion of the sample,” Hirschmugl says. “The mouse study shows that our technique has promise not only for stem cell screening without the use of dyes or probes, but also for promoting a better understanding of the biochemical structure of differentiating stem cells in their microenvironment.”
The collaborators are continuing to improve the efficiency by which they can collect and analyze FTIR spectro-microtomography with new advances being incorporated at IRENI and at IR beamline 5.4 of Berkeley Lab’s Advanced Light Source (ALS), a project being overseen by Martin. The work at the ALS is being done in collaboration with Berkeley Lab’s National Energy Research Scientific Computing Center (NERSC).
Other co-authors of the Nature Methods paper are Charlotte Dabat-Blondeau, Miriam Unger, Julia Sedlmair, Dilworth Parkinson, Hans Bechtel, Barbara Illman, Jonathan Castro, Marco Keiluweit, David Buschke, Brenda Ogle and Michael Nasse.
This research was funded by the DOE Office of Science and the National Science Foundation. Berkeley Lab’s ALS and NERSC are funded by the DOE Office of Science.
Additional Information
For more about the Advanced Light Source go here
For more about the Synchrotron Radiation Center go here

UWM discovery advances graphene-based electronics

Physics Professor Michael Weinert and engineering graduate student Haihui Pu display the atomic structure on GMO. (Photos by Alan Magayne-Roshak)

http://www5.uwm.edu/news/2012/04/13/uwm-discovery-advances-graphene-based-electronics/

By Laura L. Hunt on April 13, 2012

Scientists and engineers at the University of Wisconsin-Milwaukee (UWM) have discovered an entirely new carbon-based material that is synthesized from the “wonder kid” of the carbon family, graphene. The discovery, which the researchers are calling “graphene monoxide (GMO),” pushes carbon materials closer to ushering in next-generation electronics.

Graphene, a one-atom-thick layer of carbon that resembles a flat sheet of chicken wire at nanoscale, has the potential to revolutionize electronics because it conducts electricity much better than the gold and copper wires used in current devices. Transistors made of silicon are approaching the minimum size at which they can be effective, meaning the speed of devices will soon bottom out. Carbon materials at nanoscale could be the remedy.

Now all three characteristics of electrical conductivity – conducting, insulating and semiconducting – are found in the carbon family, offering needed compatibility for use in future electronics.

Currently, applications for graphene are limited because it’s too expensive to mass produce. Another problem is that, until now, graphene-related materials existed only as conductors or insulators.

“A major drive in the graphene research community is to make the material semiconducting so it can be used in electronic applications,” says Junhong Chen, professor of mechanical engineering and a member of the research team. “Our major contribution in this study was achieved through a chemical modification of graphene.”

GMO exhibits characteristics that will make it easier to scale up than graphene. And, like silicon in the current generation of electronics, GMO is semiconducting, necessary for controlling the electrical current in such a strong conductor as graphene. Now all three characteristics of electrical conductivity – conducting, insulating and semiconducting – are found in the carbon family, offering needed compatibility for use in future electronics.

Mixing theory and experiments

Professors Junhong Chen (left, mechanical engineering) and Marija Gajdardziska (physics) discuss the imaging with engineering graduate student Shumao Cui.

The team created GMO while conducting research into the behavior of a hybrid nanomaterial engineered by Chen that consists of carbon nanotubes (essentially, graphene rolled into a cylinder) decorated with tin oxide nanoparticles. Chen uses his hybrid material to make high-performance, energy-efficient and inexpensive sensors.

To image the hybrid material as it was sensing, he and physics professor Marija Gajdardziska used a high-resolution transmission electron microscope (HRTEM). But to explain what was happening, the pair needed to know which molecules were attaching to the nanotube surface, which were attaching to the tin oxide surface, and how they changed upon attachment.

So the pair turned to physics professor Carol Hirschmugl, who recently pioneered a method of infrared imaging (IR) that not only offers high-definition images of samples, but also renders a chemical “signature” that identifies which atoms are interacting as sensing occurs.

Chen and Gajdardziska knew they would need to look at more attachment sites than are available on the surface of a carbon nanotube. So they “unrolled” the nanotube into a sheet of graphene to achieve a larger area.

That prompted them to search for ways to make graphene from its cousin, graphene oxide (GO), an insulator that can be scaled up inexpensively. GO consists of layers of graphene stacked on top of one another in an unaligned orientation. It is the subject of much research as scientists look for cheaper ways to replicate graphene’s superior properties.

Puzzling outcome

Physics senior scientist Marvin Schofield (standing), physics doctoral student Eric Mattson, and Graduate School associate dean and physics professor Marija Gajdardziska examine the images of GMO using Selected Area Electron Diffraction (SAED) in a transmission electron microscope.

In one experiment, they heated the GO in a vacuum to reduce oxygen. Instead of being destroyed, however, the carbon and oxygen atoms in the layers of GO became aligned, transforming themselves into the “ordered,” semiconducting GMO – a carbon oxide that does not exist in nature.

It was not the result they expected.

“We thought the oxygen would go away and leave multilayered graphene, so the observation of something other than that was a surprise,” says Eric Mattson, a doctoral student of Hirschmugl’s.

At different high temperatures, the team actually produced four new materials that they collectively refer to as GMO. They captured video of the process using Selected Area Electron Diffraction (SAED) in a transmission electron microscope.

Because GMO is formed in single sheets, Gajdardziska says the material could have applications in products that involve surface catalysis. She, Hirschmugl and Chen also are exploring its use in the anode parts of lithium-ion batteries, which could make them more efficient.

Laborious process

But the next step is more science. The team will need to find out what triggered the reorganization of the material, and also what conditions would ruin the GMO’s formation.

“In the reduction process, you expect to lose oxygen,” says Michael Weinert, professor of physics and director of UWM’s Laboratory for Surface Studies. “But we actually gained more oxygen content. So we’re at a point where we’re still learning more about it.”

Weinert points out that they have only made GMO at a small scale in a lab and are not certain what they will encounter in scaling it up.

The team had to be careful in calculating how electrons flowed across GMO, he adds. Interactions that occur had to be interpreted through a painstaking process of tracking indicators of structure and then eliminating those that didn’t fit.

“It was a long process,” says Weinert, “not one of those ‘Eureka!’ moments.”

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This work was published in the journal ACS Nano (5[12], 9710-9717, 2011). In addition to the four UWM faculty members and Mattson (the lead author), the team included UWM physics research associate Marvin Schofield and postdoctoral associate Michael Nasse, UWM engineering graduate students Haihui Pu and Shumao Cui, UWM engineering research associate Ganhua Lu, and Rodney Ruoff of the University of Texas at Austin.