Fluorescent proteins have helped researchers open doors to countless molecular imaging applications and deepened our understanding of biological processes. Without fluorescence, advancements in oncology, drug discovery and any field that requires single-cell to whole-body imaging would be substantially limited.
In cancer research, for example, fluorescence imaging enables clinicians to quickly identify malignant tumor markers in blood, simplifying a once complicated process into a single step.
But fluorescent methods are not without their drawbacks. Because the technique relies on the use of external light—which can interfere with other biological processes or techniques—its application is limited.
The light required for fluorescence imaging can disrupt natural, light-dependant processes, such as photosynthesis. Also, biological techniques that rely on external light, such as optogenetics, can encounter problems with fluorescence. For these cases, an optical solution compatible with light-sensitive processes would be useful.
Chemiluminescent proteins, which generate light through a chemical reaction, offer one potential answer. However, their relatively dim light output is not on par with that of fluorescence.
The team set out to create a protein that combined the best of both chemiluminescence and fluorescence.
Creating the world’s brightest luminescent protein
At the Institute of Scientific and Industrial Research at Osaka Univ., my colleagues and I found the solution buried in the ocean sand. The sea-dwelling pansy (Renilla reniformis) compensates for a lack of light by creating its own through a process known as bioluminescence resonance energy transfer (BRET).
By fusing a luminescent protein from the sea pansy with a previously designed fluorescent protein, the team created a probe with the temporal and spatial resolution of fluorescence, all with zero dependence on external light.
The team named the new probe the “Nano-lantern.” As the world’s brightest luminescent protein, it solves the conundrum of imaging light-dependent biological processes.
In vivo imaging improvements
The Nano-lantern was put to the test in a number of proof-of-concept experiments, the first of which was in vivo imaging. In vivo studies assess biological effects on a whole, living organism, as opposed to cells or tissue in a petri dish.
Collaborating closely with Kyoto Univ.’s Dr. Yuriko Higuchi, Dr. Nagai’s team used the new probe to visualize cancer tissue inside freely moving, non–shaved mice. Living up to its promise, the Nano-lantern offered increased sensitivity and shorter exposure times compared to conventional chemiluminescent probes. To the team’s surprise, they were able to obtain video-rate imaging of tumors more than two weeks after implantation. This was a dramatic improvement over previous reports of imaging luminescent tumor cells that required nude mice, bigger tumors and longer exposure times.
In optogenetics, researchers use light to control and measure the behavior of living cells and organisms. The technique was named Nature Method’s 2010 “Method of the Year” and changed the way neuroscience studies are performed. In a recent optogenetics study, published in Neuron, the method used light to trigger emotional responses in the brain to help researchers examine the formation of anxiety and depression.
Fluorescent imaging techniques, though, aren’t always compatible with optogenetics because the same light that stimulates what's under optogenetic control can interfere with the fluorescent sensor.
To test the Nano-lantern in optogenetic imaging techniques, the team modified it into a calcium sensor and co-expressed it with a light-sensitive photoreceptor in rat neurons. They were able to follow excitation of the photoreceptors by measuring the Ca2+ increase as reported by the calcium sensor.
A photosynthesis first
The team also used the probe to study ATP production in plant chloroplasts, which occurs during photosynthesis. Using fluorescence is impossible due to the strong autofluorescence of chlorophyll and the photosensitivity of photosynthesis.
The team converted the Nano-lantern into an ATP sensor and expressed it in a live plant leaf. The Nano-lantern allowed the team to observe the increase in ATP levels after light irradiation. The team also observed what may have been the first recorded instance of a plant’s defense mechanism against high levels of light.
Imaging the Nano-lantern
To image the Nano-lantern, Dr. Nagai’s laboratory used Photometrics’ Evolve 512 EMCCD camera.
The Evolve's high quantum efficiency allowed the team to easily detect low chemiluminescent signals for both still images and video-rate imaging, without the risk of phototoxicity. The camera's superior cooling (-85 C) was particularly useful for increasing the signal-to-noise ratio necessary for imaging the mouse tumors, while its wide dynamic range enabled acquisition of both chemiluminescent and bright-field images using the same camera setting.
The Evolve also offers a feature that allowed the team to perform "cleaner" optogenetic experiments. Because the light used to stimulate optogenetic processes is so strong, it can increase the background noise level. The team took advantage of the short dead-time of the Evolve’s exposure and readout triggering cycle to conduct precisely timed optogenetic light stimulation.
Nano-lantern’s bright future
The Nano-lantern marks a giant step forward in the development of low-intensity light imaging, and can benefit applications from high-throughput drug screening to single-cell tracking in live animals and plants. With so many imaging applications made possible by the probe, the future of the Nano-lantern looks as bright as it was constructed to be.