Biologists select from many forms of imaging in an effort to answer research questions, but combining the features of different imaging methods comes in very handy in many situations. Thankfully, an increasing array of tools make such combinations more accessible. For example, new commercially available multimodal in-vitro and in-vivo systems provide a range of detection options in response to the needs of biologists. Meanwhile, commercial and academic researchers have teamed up to apply Cerenkov Luminescence Imaging to drug research, using a commercial molecular imaging platform. And a new form of microscopy combines single-molecule spectroscopy with light-sheet microscopy to enable new views of specimens. Let's take a look at how these technologies facilitate effectiveness and efficiency for biomedical research.
Looking inside cells
Most molecular biologists use many approaches for interrogating the functions inside of cells. This includes running gels and Southern blots to isolate DNA components, and Western blots to isolate proteins. Many of today's molecular biology experiments also use multiwell plates in which labels identify specific targets. To analyze such experiments, biologists use a range of imaging modalities and labels, including luminescence, fluorescence, and radioisotopes. According to Stephanie Noles, global in-vitro product manager at Carestream Molecular Imaging (Woodbridge, CT), Gel Logic Imaging Systems provide all of these imaging capabilities. For example, the Gel Logic 4000 PRO provides modes for chemiluminescence, radioisotopic, and fluorescence in the ultraviolet range. Noles adds, "Our Gel Logic 6000 PRO, the next step up, uses LED-based illumination, so researchers can use it for fluorescent imaging across the whole color spectrum: red, green, blue and near-infrared."
Noles says that Carestream combined these imaging techniques to mirror the needs of many biologists, but to also provide flexibility. "We didn't want to limit users to a particular dye or to just using DNA gels," she notes. "We also wanted to help researchers consolidate their equipment space."
She explains that the Gel Logic technology ensures that every sample receives equal illumination. "Some [systems] use a zoom lens to focus on different samples," says Noles, "but ours actually moves the sample closer or farther from the camera for focus. In addition our light sources are mounted to the sample platform. That way, every sample gets the same illumination and light scatter is minimized."
All Gel Logic Systems also use the same software, which includes a collection of one-step protocols for common needs, like analyzing a DNA gel, blot, or plate. For the Gel Logic 6000 PRO, researchers can even simultaneously track up to four different labels. In addition, Noles notes that modularity makes it possible to upgrade as needed. "As your research needs change," she says, "your instrument can evolve."
Merging modes
Medical research often uses optical systems that pair light and nuclear approaches, including positron emission tomography (PET), which images radiation. Cerenkov Luminescence Imaging (CLI) is a technology that combines these approaches. In PET, researchers use radioactive labels that emit high-energy, positively charged particles called positrons. As these particles pass through a tissue, they create tiny bursts of light—which Robbie Robertson, a scientist in the biomedical imaging group at Millennium: The Takeda Oncology Company, thought modern imaging techniques might be able to see.
In 2007, Robertson started working on CLI. Working with Simon Cherry, professor of biomedical engineering at the University of California, Davis, Robertson reported in 2009 that CLI can be used in live animals to follow molecular markers. "You can use the atomic light to spark fluorescent probes," Robertson explains. So this technique can combine radio- and fluorescent labeling. For example, one type of label could be used to locate specific receptors and another to tag a compound that might inhibit or activate that receptor. "This has the ability to really increase the throughput of drug discovery," Robertson says. Compared to using PET, for instance, Robertson and his colleagues scanned animals 10-fold faster with CLI.
Using the IVIS three-dimensional imaging platform from Caliper (Hopkinton, MA), Robertson and his colleagues at Millennium use CLI to, as he says, "examine the distribution of experimental compounds, so we can see how the organ levels change." He adds, "We're looking into using it at the very beginning of developing a drug."
Some commercial systems also offer a range of imaging modalities from one platform. For example, Seth Gammon, in-vivo product manager at Carestream Molecular Imaging, says, "Our Xtreme combines high-sensitivity luminescence, fluorescence, radiographic, and radioisotopic imaging." Given this range of imaging modalities, he points out that the Xtreme can be used to "study different pathways or reporters in small animals." He adds, "New probes and biomarkers keep coming out, and this platform will let scientists start using them without needing additional equipment." In addition, the Xtreme's software co-registers the images from different modalities to make one composite image. Researchers can even buy this platform with what Gammon describes as "a choice of cameras to match their budget to their application and upgrade later."
Combining techniques
Working with single-molecule spectroscopy, says Malte Wachsmuth, staff scientist in the Cell Biology and Biophysics Unit at the European Molecular Biology Laboratory (Heidelberg, Germany), provides a powerful method to explore molecular interactions and mobilities in living cells and tissues, but he wanted to see even more. He wanted to track the fluorescence from single molecules across an entire sample.
"For any single-molecule technique," says Wachsmuth, "you need really good background suppression and three-dimensional resolution to get the signal from a single molecule and distinguish that from the background, which depends on the optics." To collect the fluorescence from lots of single molecules in parallel, Wachsmuth incorporated it with light-sheet microscopy, which uses a plane of light to illuminate a sample in slices (see figure). "This provides an array of confocal observation points," says Wachsmuth.
Wachsmuth wanted to use this technology to track targets in the nucleus, but he found that the system did not allow him to distinguish particles well enough to track them. So he emulated correlational microscopy, which he says "looks at fluctuations of particle concentrations at a single spot. From that, you can learn about the speed of diffusion of molecules."
To make this work, though, Wachsmuth and his colleagues built the system themselves. "We use Zemax optical-modeling software to establish the optics, then CAD software for the 3D construction of the pieces we cannot buy," he says. "Then, we build it. After that, we write software that controls the motors and the camera." His team also developed the data-processing software. "It's pretty much nonstandard," Wachsmuth says, "so we wrote it."
With so many steps to complete, it took Wachsmuth and his colleagues about a year and a half to start getting reasonable data. "It took another year to get really decent data," Wachsmuth says.
With this platform up and running, though, Wachsmuth and his team can see many new things. "We can quantify molecular interactions in a spatial manner that also provides good time resolution," Wachsmuth says.
By combining multiple approaches, researchers are able to explore previously unseen aspects of biology. The results show the value of multiples: Combining different imaging techniques in some cases, and merging the features of more than one technique in other situations.