Angelo DePalma earned his Ph.D. in organic chemistry from Stony Brook University and was previously senior scientist at Schering-Plough. He has written extensively on biotechnology, biomanufacturing, medical devices, pharmaceutical commerce, laboratory instrumentation, and advanced materials.
Acquiring images in real time from living organisms has become indispensable in too many life-science disciplines to mention. In just the last few weeks, we’ve seen notable advances in molecular and small-animal imaging, including:
- Research from Duke University on high-resolution mouse neuronal modeling based on the “connectome” or connection circuitry within a mouse’s brain. Investigators claim resolution that is 100,000 times higher than that obtained by clinical-grade magnetic resonance imaging.
- A Harvard University group that uses stimulated Raman scattering (SRS) microscopy to image DNA dynamics during cell division. Label-free SRS measures the vibrational frequencies of chemical bonds, which change based on the surrounding molecular environment.
- The debut of the cellFRAP imaging system by Olympus. FRAP (fluorescence imaging after photobleaching) is a microscopic technique available from several vendors. Differentiating the cellFrap system is its ability to image an entire field of view without moving the sample.
- The introduction of UVP’s iBox Scientia 900 In Vivo Imaging System for detecting fluorescent and bioluminescent markers in small animals.
- A breakthrough from Bruker Biospin for imaging inflammation in small-animal disease models through multimodal imaging using appropriate combinations of bioluminescence, fluorescence, radioisotopic and X-ray imaging. Bruker has also reported the application of its MS FX PRO imaging system for visualizing ongoing infection in animals through multimode imaging.
- NKT Photonics’ description of ultrahigh-resolution optical coherence tomography (OCT), which enables micron-scale, cross-sectional and 3D-imaging capabilities in live animals. OCT is based on white-light interferometry, in which light reflected from a sample interferes with light from a reference arm.
Mouse to man?
Vivek R. Shinde Patil, senior manager of technical applications for preclinical imaging at Revvity, calls 2015 a significant year for his company. This marks the twentieth anniversary of a seminal paper by Contag, et al., that demonstrated the feasibility of noninvasively imaging bioluminescent reporters in small animals [1]. “That set the stage for development of the field,” Patil says. From that study emerged Xenogen, which introduced bioluminescence-based imaging platforms that laid the foundation for more advanced IVIS® imaging systems. Xenogen was then acquired by Caliper Life Sciences, which later developed more sophisticated fluorescence imaging, spectral unmixing and 3D optical tomography. We came full circle with Revvity’s 2011 acquisition of Visen and its 3D fluorescence technology and reagents, and finally its purchase in 2012 of Caliper Life Sciences. Today Revvity possesses a comprehensive portfolio of in vivo imaging tools based on bioluminescence, fluorescence, microCT (computerized tomography) and radioisotopic methods.
Patil estimates the number of publications based on Revvity imaging platforms at greater than 6,000. “Users of our technologies publish seven to eight hundred papers per year, validating the widespread adoption and application of Revvity technologies and reagents.”
In addition to offering 2D and 3D optical imaging, Revvity has introduced a microCT system. “Optical [imaging] is highly sensitive and provides molecular and functional information but offers limited resolution, because light diffuses through tissue,” Patil explains. “Our preclinical microCT provides the highest-resolution anatomical visualization on the market.” The company also has partnered with Sofie Biosciences for a benchtop PET system that relies on radioisotopes and, like CT and optical imaging, provides translational medical opportunities.
According to Patil, multimodal, complementary in vivo imaging and translational applications are exciting trends. His firm’s IVIS SpectrumCT, for example, encapsulates that concept by combining 3D bioluminescence, fluorescence, and microCT in one platform. And although the use of widespread optical methods in human diagnostics is still far off, he believes these methodologies will eventually lead to a unified “mouse-to-man” imaging paradigm.
Top-of-the-line multimode imaging systems with super-high resolution cost a million dollars or more. Multimode optical imagers occupy a market that is much more price-sensitive, costing $150,000 to $200,000. Also square within the lower price category are complementary technologies such as X-ray transmission imaging, which generates a map of the animal’s skeleton, thereby precisely localizing features and characteristics.
Complement, not replace
The in vivo imaging business of LI-COR Biosciences is based on bioluminescence and fluorescence-based optical technology. The company offers both instrumentation and near-infrared (NIR) dyes. “Most fluorescent in vivo imaging occurs in the near-infrared because of absorption of light in tissues,” says Jeff Harford, LI-COR senior product and marketing manager. LI-COR got into the imaging business through its original specialty, which was NIR dyes and instruments. LI-COR’s fluorescent dyes are particularly suited to in vivo work because of their clearance properties, and they are easily attached to biomolecules. LI-COR’s imaging system, the Pearl Trilogy Small Animal Imaging System, operates through near-infrared fluorescence and bioluminescent optical imaging. One of the company’s fluorescent dyes is currently in human clinical trials for cancer visualization, and a second dye is in human clinical trials for immunotherapy.
According to Harford, the large imaging user base has reached a critical juncture, and lab managers are willing to consider replacing aging equipment. This is in part because of the growing preference for fluorescence over bioluminescence, particularly for translational research. Moreover, newer instruments are smaller and less expensive than older models, and they have greater functionality. “The effect of technologic and scientific trends is that small-animal in vivo imaging systems can move out of core facilities to satellite labs,” Harford says. Simplicity and ease of use also drive the democratization of imaging, as labs increasingly employ relatively more technician-level workers than degreed scientists.
Harford recently attended the 2015 World Molecular Imaging Conference, which devoted an entire day to surgical-optical navigation, a field heavily reliant on the small-animal fluorescence work currently under way. A prominent theme of the conference’s workshops was the translation of ongoing successes to the clinic. “This, I believe, will be the focus of in vivo imaging work over the next five years,” states Harford. Further development and improvements in infrared-laser-based devices for excitation and signal capture will be critical, both in creating robust handheld systems and vis-à-vis the inevitable regulatory issues.
Harford concedes that fluorescence imaging is unsuitable for noninvasive disease detection. Its primary applications will be for tumors that are close to the skin surface or to guide surgical resection (e.g., determination of tumor margins). “The imaging applications will complement, not replace, traditional imaging modalities,” Harford notes. An offshoot of this idea is the potential use of targeted photodynamic therapy using dyes attached to targeting molecules, or photoimmunotherapy that involves attaching dyes to antibodies.
Plant imaging has become common in crop science and agribusiness. Because gravity affects critical events in plants, imaging systems should have the capability to view from the top and sides. “Light in imaging chambers should also be precisely controlled for intensity and spectral distribution to simulate day/night cycles [and] circadian rhythms and to activate or deactivate the plant’s photoreceptors,” says Dr. Frank Schleifenbaum, head of product management and marketing at Berthold Technologies in Germany.
The tools and lexicon of plant imaging are similar to those for small animals: fluorescence and chemiluminescence; dyes that accumulate in certain tissues; and the up-regulation of genes responsible for protein expression and life-cycle events. “Pretty much whatever you can do with animals, you can do with plants,” Schleifenbaum says.
What sets plants apart from mammals, however, is that plants’ adaptation mechanisms are much more complex. “The reason is that plants cannot run away,” Schleifenbaum explains. “They must accept the conditions they face and adapt to more or less light, warmth or cold, more or less nutrition, without the ability to protect themselves.” It is in fact plants’ coping processes that drive much of plant-based imaging.
A key goal of this work is improved yield, particularly but not exclusively in genetically modified crops. Researchers, Schleifenbaum says, are eager to learn how growth and seedling behavior depends, for example, on light quality, and how plant life cycles are organized—and eventually to modify these events to advantage. “In vivo imaging allows investigators to reach these understandings noninvasively, without having to extract proteins and with minimal impact on the living organism.”
Reference
[1] Contag, CH, et al., “Photonic detection of bacterial pathogens in living hosts,” Mol Microbiol, 18:593-603, 1995. [PMID:8817482]
Image: www.research.usc.edu