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New Biological Frontiers Illuminated by Molecular Sensors and Actuators
Monday Speaker Abstracts
Revolutionary Bioimaging with Bright Luminescent Proteins - Comparing Pros and Cons
of Fluorescence and Luminescence
Takeharu Nagai
.
Osaka University, Osaka, Japan.
Fluorescent proteins is an indispensable tool for live imaging of cells and cell structures. But the
requirement for external illumination definitely precludes its universal application because it can
cause problems including photobleaching, photodamage and the unintended activation of other
light-responsive proteins. Luminescent proteins such as luciferase is an alternative to
fluorescence that does not require an excitation light. However, luminescence imaging has been
limited by the dim brightness and lack of color variation of existing luminescent proteins. To
overcome this drawback, we conducted random mutagenesis on Renilla reniformis luciferase
(Rluc) gene to improve the intensity. Then, the luminescence intensity was further increased by
fusion of the improved Rluc to a yellow fluorescent protein Venus with a high BRET efficiency.
The chimeric protein showed much brighter luminescence than the original Rluc, enabling not
only real-time imaging of intracellular structures in living cells with spatial resolution equivalent
to fluorescence but also sensitive tumor detection in freely moving mice which has never been
possible before. We also developed color variants of the Nano-lantern by substitution of the
Venus with a different wavelength fluorescent protein. Furthermore we applied these Nano-
lanterns to design Ca2+, cAMP, and ATP indicators, thereby we succeeded imaging these
bioactive molecules in environments where fluorescent indicators have failed. These luminescent
proteins will revolutionize conventional bioimaging by allowing visualization of biological
phenomena not seen before at the single-cell, organ, and whole-body level, in animals and plants.
While luminescence may be a practical alternative in situations where fluorescence is
problematic, the signal intensity of the Nano-lanterns is still more than 100 times weaker than
fluorescent proteins. In the symposium, I will compare pros and cons of fluorescence and
luminescence for bioimaging.