An inner light

technology feature An inner light In vivo bioluminescence imaging offers a non-invasive look inside the body. Its future looks bright. Michael Eisenstein S hinae Kizaka-Kondoh recently had the opportunity to read a monkey’s mind. Using an imaging system called ‘AkaBLI’, Kondoh—a researcher at the Tokyo Institute of Technology—and colleagues were able to observe neurons firing within the brain of a marmoset as it roamed about its cage. Traditional methods of looking at the cellular and molecular activity of the brain generally entail restraint or anesthesia, or require surgery or dissection. AkaBLI is a newly developed iteration of a non-invasive method called bioluminescence imaging (BLI), and it allowed the researchers to visualize brain function through the intact skull of a freely moving animal over the course of several months1. “It is wonderful to be able to get such information from inside the body with light,” Kondoh says. Scientists have been exploiting bioluminescence—the enzymatic reaction that produces the distinctive glow of animals like the firefly—as a visual readout of biological activities in cultured cells for decades. But more recently, researchers have come to recognize it as a simple, non-intrusive method for direct in vivo imaging in animal models. Newcomers are routinely taken aback by what they can observe with BLI. Gary Luker of the University of Michigan recalls showing a collaborator imaging data from mice that had been infected with bioluminescent herpesvirus back in 20022. “I still remember his amazement to actually see the site of infection,” says Luker. “In some immunocompromised strains, we could see that an infection that started in the footpad of the mouse may have spread to the central nervous system, which nobody really had even thought about before.” With an expanding toolbox of reagents and increasingly sophisticated detection equipment, in vivo bioluminescence imaging (BLI) has become a useful tool for researchers studying dynamic processes related to cancer and infectious disease. And as high-profile demonstrations like the recent work from Japan continue to emerge, many early adopters believe BLI is ready to go mainstream, offering life scientists an accessible window into the inner workings of the body. From nature to the lab: Perhaps the best known bioluminescent animal is the firefly, which has lent its glow to the lab. Other sources—natural and engineered—are improving bioluminescence imaging too. Credit: tomosang / Getty Penetrating analyses When it comes to bioimaging, fluorescent proteins generally hog the glory. These molecules do not shine on their own, but emit a bright signal when illuminated at a particular wavelength. Extensive efforts towards fluorescent protein discovery and engineering have produced a diverse palette of molecules that produce colors spanning the spectrum. By coupling these to different genes, one can visualize molecular-scale processes within cells, or use combinations of fluorescent proteins to track many cellular targets at once. In contrast, bioluminescence is produced by a chemical reaction mediated by enzymes known as luciferases. In naturally bioluminescent animals, luciferase processes its substrate in a chemical reaction that results in the emission of photons, generating visible light. The firefly Photinus pyralis is perhaps the best-known example. Its greenish-yellow glow comes from the reaction between luciferase and a substrate molecule called d-luciferin. However, there are a host of other bioluminescent insects, including various beetle species, and numerous marine organisms ranging from plankton to fish Lab Animal | VOL 47 | NOVEMBER 2018 | 301–304 | www.nature.com/laban that also use luciferase-mediated reactions to generate light. Over the past three decades, molecular biologists have identified and cloned a number of different luciferase genes. For in vivo BLI, animals—in most cases, mice—are genetically modified to express one of these genes. The objective is for the Tracking tumors: The Luker lab uses bioluminescence imaging to monitor tumor progression. Credit: G. Luker 301 technology feature animal to produce the luciferase enzyme in response to a particular biological event, such as the expression of a specific gene or activation of a particular cellular protein, or in a particular subset of cells. The strategy used for genetic modification is a critical component of this process, and can profoundly affect the outcome of the experiment (see Box 1). After the animal has been administered the enzyme’s substrate, it will produce a measurable glow at the time and place where luciferase expression occurs—typically within a matter of seconds or minutes of the gene being switched on. The fact that BLI does not require external illumination gives it an edge over fluorescence for some in vivo applications. The laser light used to excite fluorescent proteins generates considerable unwanted background glow as it illuminates and scatters off tissues, making it difficult to decipher imaging data. “It’s not so much that the signal from bioluminescence is higher,” explains Jennifer Prescher of the University of California at Irvine, “it’s that the background noise is almost zero.” If a researcher sees light with BLI, it’s probably from a true biological event. BLI can also image deeper inside the living animal, enabling a view of tissues where the excitation light required for fluorescence would be too heavily scattered to generate useful data. In practice, this means that researchers can look several centimeters beneath the skin with BLI, whereas such a feat with fluorescence would require surgery to give the microscope a clearer view. “There’s a massive emphasis now in the UK on the ‘three Rs’ and improving animal welfare,” says Simon Waddington at University College London (UCL). “This non-invasive imaging plays right into that.” Animals can be routinely monitored with BLI for days, weeks or even months. Researchers have been obtaining intriguing data from in vivo BLI since the first commercial imaging instruments hit the market in the early 2000s. Early studies mainly entailed tracking labeled cells, bacteria or viruses within the rodent body, 302 that are very sensitive to photons, so you can get millisecond-scale acquisition of the image while the animal moves,” says Laura Mezzanotte of the Erasmus Medical Center. This can be invaluable for studies of metabolic activity, which would be confounded by the physiological changes experienced by an immobilized or anesthetized rodent. Rajvinder Karda, a researcher studying gene therapy at UCL, notes that her studies of central nervous system function have benefited immensely from the elimination of anesthesia. “Isoflurane can dampen the development of the growing brain, and we are trying to monitor luciferase expression at neonatal stages of development,” she says. “We’re exposing them to less chemicals and it’s a very quick (...truncated)