How the world’s brightest luminescent protein is lighting up the future of in vivo imaging without the use of external light

Fluorescent proteins offer researchers a window into the complexities of biological processes, making advancements in high-throughput drug screening and other applications possible.

Researchers using fluorescence can sometimes encounter challenges, though, because of the technique’s dependence on external illumination. For instance, the light needed to activate fluorescence can disrupt other biological processes, including photoreception or photosynthesis. Similarly, in the emerging field of optogenetics, the same light that excites fluorescence also triggers the optogenetic reaction. In such cases, it would be ideal to uncouple these processes so that each could be studied separately.

Chemiluminescence– generating light through a chemical reaction– offers one solution to this problem. But, the light output of most chemiluminescent probes is too dim to serve as a viable fluorescent alternative.

An internal struggle

The challenges associated with fluorescence are particularly evident when researchers employ in vivo imaging techniques. In vivo imaging allows researchers to study cellular events as they occur in the body– offering the most relevant insight. However, these events often occur in deep tissue far below the skin, where light cannot accurately penetrate.

Fluorescence can offer one solution for in vivo imaging, but image quality suffers as photons from the external light source scatter through tissue on their way to the fluorescent target. It’s similar to the effect of shining a laser pointer through your thumb– the light will come out the other end, but not as the single beam at which it started.

Without a reliance on external light, a chemiluminescent approach would not suffer these drawbacks, but the light generated would be too weak to allow for high-quality imaging.

What researchers need, then, is a chemiluminescent solution that’s as bright as fluorescence.

A bright idea

The first place researchers looked to find this solution was nature itself. Luminous organisms, such as the sea pansy (Renilla reniformis) compensate for a lack of light in their environments by creating it themselves through a process known as bioluminescence resonance energy transfer (BRET).

Researchers are taking advantage of this process to develop probes that can help improve in vivo and other imaging. In 2012, researchers at the Institute of Scientific and Industrial Research at Osaka University engineered a probe that fused a luminescent protein from the sea pansy with a previously designed fluorescent protein.

The result—dubbed the “Nano-lantern” by its creators— is the world’s brightest luminescent protein, possessing the temporal and spatial resolution equivalent to fluorescence – all with zero dependence on external light.

Improved in vivo

With the Nano-lantern’s improved image quality and brightness, the protein was put to the test in several feasibility experiments, which included in vivo imaging studies.

In a study visualizing cancer tissue inside freely moving, non-shaved mice, the Osaka University researchers worked with Kyoto University’s Dr. Yuriko Higuchi to uncover some distinct advantages the Nano-lantern offered over conventional probes. In addition to increased sensitivity and shorter exposure times, the Nano-lantern allowed for video-rate imaging of tumors 17 days after implantation.

The increased sensitivity and faster imaging led to a greatly improved analysis of tumor growth in non-anesthetized, living animals. In previous luminescent tumor cell imaging studies with non-anesthetized mice, successful images were obtained only with nude, shaved mice and larger tumors.

Taking the picture

To capture the full imaging benefits of the Nano-lantern, researchers used the Evolve 512 EMCCD camera from Photometrics.

Several key features of the Evolve 512 camera made it an ideal choice for working with the Nano-lantern. The camera’s high quantum efficiency enabled the detection of low chemiluminescent signals for both still images and video-rate imaging without the risk of phototoxicity. Signal-to-noise ratio for the mouse tumor imaging was increased due to the camera’s superior cooling (-85°C). In addition, the Evolve’s wide dynamic range allowed researchers to obtain both chemiluminescent and bright-field images with the same camera setting.

The future of Nano-lantern

Researchers are already hard at work creating new colors for the Nano-lantern and boosting its brightness, with a goal of enabling single-molecule-level imaging. There are no plans to commercialize the Nano-lantern and the DNA construct is freely available– meaning more biologists can benefit from its brightness.

Touting increased brightness and expanded capabilities, the Nano-lantern could facilitate more advanced applications, such as high-throughput drug screening and single-cell tracking in live animals and plants. It could also help realize the other advantages of low-intensity light imaging, including diminished photobleaching and phototoxicity, which allow for prolonged observation without harming the specimen.

With an eye on the future, researchers are exploring ways to improve the Nano-lantern. Currently, rapid consumption of the luminescent substrate by Nano-lantern hampers prolonged observation of biological events, especially in the case of whole-body imaging. To address this issue, researchers are working to develop a method that facilitates indefinite observation applicable to the entire organism. Nevertheless, it does not diminish one very bright accomplishment.