Multiple views, better data from a single tool

Integrated tools empower multidisciplinary collaboration by accommodating diverse tasks

By Sarah Fister Gale

When Marc Fries Ph.D. studies the makeup of ancient meteorites that were formed before the planets, he uses three-dimensional confocal imaging to see how inclusions in primordial silicate droplets are arranged: Raman spectroscopic imaging to identify key minerals, and atomic force microscopy to study features too small for optical measurement techniques. And he does it all simultaneously, without ever moving his sample.

Fries, a research scientist at the Geophysical Laboratory at the Carnegie Institution of Washington, D.C., uses a WITec Alpha 300 Scanning Near-field Optical Microscope (SNOM), which offers multiple imaging options on a single piece of equipment by rotating the objective turret. The WITec model includes a confocal microscope, which employs spatial filtering to produce images that are free from out-of-focus blur; a SNOM, which uses a small probe to scan across the surface and build up an image line-by-line; a Raman spectroscopic imager that creates maps of mineral phases; and an atomic force microscope (AFM), which delivers demonstrated resolution of fractions of a nanometer, more than 1,000 times better than the optical diffraction limit.

By combining these multiple views, Fries can gather more-complex data from his samples with a smaller margin of error. “Whenever you can use multiple complementary technologies on a single sample, it improves the confidence you have in your data,” he says.

HeLa cells depicted in an AFM amplitude image, 40μm scan (left, top) and a TIRF image, 40μm scan (left, bottom). At right, the TIRF data is overlaid on the rendered AFM topography. (Image courtesy of M. Kellermayer, University of Pécs; and Asylum Research)
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Because the sample does not need to be moved, Fries can examine the same section of the sample using multiple imaging techniques, which eliminates discrepancies in his research that result from transporting the sample among stand-alone pieces of equipment. “You can take measurements on the exact same spot making the data easier to correlate and more publishable,” he says.

That consistency is just one of the reasons multiple imaging tools, like the Alpha 300, are gaining popularity among cutting-edge researchers. While not often used in commercial labs, academic researchers studying surface and materials science, geosciences, life science, pharmaceutical development, thin films and coating analysis, nanophotonics, and nanotechnology are quickly discovering the potential that merged microscopy tools deliver to their work.

Tools follow trends

“Integrated microscopy is like a Swiss Army knife for developmental technology,” says Terrence Lundy, vice president and managing director of Hyphenated Systems, a maker of advanced hybrid microscopy solutions for research and industrial applications in Burlingame, Calif. “It’s the ultimate in flexibility for multidisciplinary tasks.”

These tools add convenience and reliability to the process while cutting costs and space constraints, because facilities can invest in a single piece of equipment to achieve multiple tasks. But they also support the larger trend in fields where multidisciplinary research facilities and projects are becoming the norm, and researchers must work with experts from outside their peer groups.

Forward-looking high-perfor­mance microscope manufacturers, such as WITec, Hyphenated Systems, Veeco, Asylum Research, Agilent, and others, recognize that multidisciplinary work is becoming an integral part of new research and are responding with more-flexible microscopy tools. “The trend toward multidisciplinary tasks is growing,” Lundy says, “and these tools facilitate multiple researchers’ needs.”

Development work on innovative systems that combine technologies-such as microfluidics, lab-on-a-chip, and biomedical devices-require the coordinated efforts of engineers and biotechnologists working on a single wafer or device. By combining multiple imaging techniques into one tool, researchers from different disciplines can work in the same space with the same equipment, at a lower cost, accomplishing multiple tasks simultaneously.

Three-dimensional point of view

Carlos Hidrovo, research associate at the Stanford Microfluidics Laboratory in Palo Alto, Calif., is taking advantage of integrated microscopy in his work on microfluidics, using Hyphenated Systems’ 3D Map to study vapor flow and microparticle velocity in microchannels. 3D Map combines optical profiling and imaging quality with micron-resolution particle image velocimetry (micro PIV) capabilities in a single platform to characterize structure and flow in microfluidic devices.

“To capture the relevant information about moving liquid structures we need to be able to get fast three-dimensional images on multiple vertical planes; otherwise the images are blurred,” he says.

For the structural metrology, the confocal microscopy permits fast, accurate 3D modeling of top surface and subsurface features in operating devices. For flow measurement, micro PIV refines the capabilities of existing techniques, such as particle image velocimetry (PIV), by resolving data in depth as well as laterally.

“Using 3D Map, the confocal imaging shows us the material while the PIV measures the liquid flow so we can understand more factors of the flow,” according to Hidrovo. It has also enabled the team to study surface tension at the micro scale, in addition to the temperature and pH balance of the flow.

“It’s like getting a key to a magic room,” Hidrovo says of having access to the 3D data. “We don’t know what we will find, but we know we are going to learn a lot. We have the ability to achieve a new understanding of physical awareness. We’ve never looked at fluids at this scale before.”


The combination of imaging technologies lets researchers gather data not previously accessible using the tools in isolation, adds Sebastian Tille, senior life science product marketing manager for Woodbury, N.Y.-based Veeco, a provider of instrumentation to the nanoscience community. Veeco recently partnered with Leica Microsystems GmbH, a designer and manufacturer of optical microscopy imaging systems in Wetzlar, Germany, to combine Veeco’s BioScope II AFM with Leica’s DMI series of inverted microscopes to offer multiple high-resolution imaging options for cell biology (and thus, says Veeco, establishing compatibility with all commercially available light microscope research platforms). The tool allows researchers to correlate fluorescence images with topography for more insight into nanostructures and functions of cells and perform nano-mechanical measurements such as elasticity, molecular unfolding, and ligand-receptor interactions.

“AFM is a powerful complement to light microscopy, and it is enabling researchers to focus down to the atomic structure,” Tille says. “Moreover, they can ‘feel’ the sample rather than just look at it. With that sense of touch, they can measure properties like adhesion or stiffness of living cells, adding an entire new dimension for scientists.”

This topographic AFM image shows the surface of a polymer blend (PMMA/SBR) spin-coated on glass at 20 x 20μm. It reveals round features, 20 to 30nm in height, with a diameter from 150nm to 4μm.
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The AFM in combination with other microscopy tools can also be used to stimulate activity in a sample, notes Dr. Irene Revenko, biologist for Asylum Research, in Santa Barbara, Calif., which offers the MFP-3D-CF integrated confocal and AFM system. For example, if biologists want to study calcium in cells, they can use the AFM probe to prod the cell causing calcium flow, which they can simultaneously track using high-resolution optical imaging. “It’s beautiful the way you can follow the flow. It’s like taking a movie,” she says of the process.

These technologies aren’t new, but they are improving, she adds, noting that AFM technology in particular is steadily getting more sophisticated. Until recently it was used only for dry samples, but now the technology can be used with liquid samples, including cells, which is drawing the interest of scientists who didn’t previously consider the value of AFM tools. “As biologists become more aware of how they can use AFM technology in combination with other imaging techniques, such as optical fluorescence and confocal microscopes, more-interesting data will result,” she says. “These tools could greatly improve research in the bioscience community.”

Color-coded confocal Raman images of the same sample area show the distribution of PMMA (red) and SBR (blue), clearly indicates phase separation in the two polymers. Comparing the data to the AFM image (left) reveals that the higher, round features are PMMA down to the glass substrate, whereas the 30nm-high background represents SBR phase. (Images courtesy of WITec)
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It also seems clear that as researchers continue to embrace multidisciplinary projects that require exploration at the nanoscale, integrated microscopy tools will become integral to their work. “A lot of researchers want minimum fuss to do a wide variety of measurements,” says Fries. “Someday maybe one instrument will have the capacity to do it all.”


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