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Imaging The World’s Brightest Luminescent Protein With Photometrics Evolve

Optogenetics was named as ‘Method of the Year’ by Nature Methods in 2010. This technique utilizes both optics and genetics to control and examine the activity of individual neurons in living tissue.

Today, laboratories worldwide are using this technique to examine everything from neurodegenerative diseases to decision making, often with remarkable results.

Stimulating, but interfering

As an example, researchers have demonstrated that light can be used to steady the stumbling gait of a rat with Parkinson’s Disease. Fluorescent imaging plays a fundamental role in examining biological function, but problems arise in techniques such as optogenetics where external illumination is required. The same light which is used to stimulate neurons under optogentic control can interfere with the fluorescent probes. For example, blue light which excites FRET-based indicators in calcium imaging also activates a photo-sensitive receptor which is commonly used in optogenetics.

Light can also be generated by chemiluminescence, but existing probes are too weak for optogenetic studies.

Meeting the challenge

The challenge was taken up by Professor Takeharu Nagai from Osaka University, an inventor of numerous ingenious fluorescent and chemiluminescent probes. He wanted to develop a probe which was compatible with optogenetic studies, in part because he had research questions of his own which he wanted to answer.

“To investigate the input-output relation in living cells, compatible use of optogenetics and fluorescent indicators, such as calcium ion probes, are very useful combinations. However, because fluorescent indicators require an excitation light source, such excitation light gives perturbations to the cell function as an additional source of photo-stimulation light”.

Behold the Nano-Lantern!

To overcome this problem, Prof Nagai’s lab engineered a new probe- the ‘Nano-Lantern’! This probe fuses a luminescent protein from the sea pansy (Renilla reniformis) with a previously designed fluorescent protein. The result was the world’s brightest luminescent protein which had the temporal and spatial resolution equivalent to fluorescence.

Once they had designed the new, considerably improved protein (ten times brighter than the original RLuc template), they tested it in living cells. Firstly, they used it to image intracellular structures and demonstrated spatial resolution and brightness which was equivalent to that of fluorescence.

Observing cancer and calcium

In a collaborative project with Dr. Yuriko Higuchi at Kyoto University, they visualized cancer tissue in a live and freely moving mouse. In these experiments, the Nano-lantern offered many advantages over conventional probes, including shorter exposure times and increased sensitivity. The team were able to image tumours 17 days after implantation.

The next achievement for Prof Nagai and his group was to modify the Nano-lantern into a calcium sensor and co-express it with a photoreceptor in rat neurons. Fluorescent calcium indicators are triggered by light, which means they often cannot be used in optogenetics. However, the co-expression system allowed the group to examine the excitation of the optogenetic photoreceptors by measuring calcium increase as indicated by the Nano-lantern calcium sensor.

Cutting edge imaging for cutting edge science- the Evolve 512 EMCCD

To visualize the Nano-lantern signals highlighted above, Prof Nagai and his colleagues used the Photometrics Evolve 512 EMCCD camera. Prof Nagai said “We knew Photometrics cameras worked well because we had used the Photometrics Cascade II EMCCD”. Prof Nagai has been using Photometrics cameras for more than a decade, and this technology has contributed to 11 of his publications.

The high quantum efficiency of the Evolve meant that the team could easily detect the low chemiluminescent signals of RLuc in both still images and video-rate imaging- without risking phototoxicity. The superior cooling of this camera (-850C) increases the signal-to-noise ratio in the mouse tumor imaging, and its’ wide dynamic range enables the acquisition of bright-field and chemiluminescent images with the same settings.

 

Luminescence (left) and fluorescence (right) of HeLa cells expressing the Nano-lantern probe targeted to the mitochondria, cytoplasm and Histone 2B. The exposure times for the luminescent images were 3 secs, 3 secs and 1 sec. The exposure for the fluorescent images were all 1 sec. Scale bars are 50 µm.

Cleaner optogenetics thanks to the Evolve Camera

Prof Nagai and his team were able to perform ‘cleaner’ optogenetic experiments due to a feature of the Evolve 512 EMCCD camera. The stimulation light used in these experiments is so strong it can increase the background noise. “One of the unique advantages of the Evolve 512 is that it can erase the charges on the CCD during the dead-time,” said Prof Nagai. “Now all biological phenomena that are regulated by light, including photosynthesis and photoreception in the retina, can be viewed using Nano-lantern, giving us more dynamic and quantitative analysis of living cells”.

A bright future for the Nano-Lantern

The team at Osaka University are already working on ways to boost the brightness and create new colors for the Nano-lantern; it is hoped this could lead to single molecule-level imaging. There are currently no plans to commercialize the Nano-lantern; instead, Prof Nagai is making the DNA construct freely available. With increased brightness, it is expected that the Nano-lantern will allow more advanced applications such as single-cell tracking in live animals.

Finally, Prof Nagai notes that prolonged observation of biological events is hampered by the rapid consumption of the luminescent substrate by Nano-lantern. However, the team are busy working towards a method which will allow continuous and indefinite observation, even in entire organisms.

For more information on this research, to view videos from the work of Prof Nagai’s team and for information on the Photometrics Evolve camera, please click here.

 

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