Positively photoswitchable fluorescent proteins with high photostability and photoswitching speed

Key Word : Kohinoor, fluorescent protein, super-resolution microscope

Background / Context / Abstract:

Reversibly photoswitchable fluorescent proteins (RSFPs) have a crucial role in nanoscopy imaging such as PALM, NL-SIM, pcSOFI, and RESOLFT. Among the available RSFPs, most of them categorized in negatively photoswitchable RSFP (NS-RSFP), in which the excitation light induces the transition from an on-to-off state. However, NS-RSFPs have limited application due to gradual switching off during imaging. Meanwhile, positively switchable RSFP (PS-RSFP) can be switched-on upon fluorescence excitation light. This property is especially favorable for nanoscopy imaging as it reduces the image acquisition time and excitation light power.
Here the inventors developed the novel PS-RSFP, termed “Kohinoor,” meaning an old famous diamond found in India, which possesses superior photoswitching speed as well as photostablity.

Technology Overview:

・Positively photoswitchable fluorescent proteins; switched on by a light used for fluorescence excitation.
・Superior photoswitching speed.
・High photostability and fatigue resistance over many switching cycles.
・Less phototoxity; extremely low laser power for RESOLFT nanoscopy.

Benefits:

Comparison between this technology “Kohinoor” and a prior PS-RSFP “Padron” in terms of photoswitching speed and photostability.
The speed for switch-on and switch-off kinetics of Kohinoor was ~4 times and ~3 times faster than Padron, respectively.
Kohinoor showed a very slow reduction in fluorescence intensity of ~0.1% in each consecutive switching cycle, whereas Padron showed 2.5% reduction. Kohinoor’s rapid on/off switching and superior fatigue resistance allowed it to achieve 25 times more switching cycles than Padron before its fluorescence decreased to 50% of its initial intensity.
Kohinoor can be fused with various fusion proteins (vimentin, clathrin, histone, paxillin, actin, zyxin, mitochondria, golgi, tubulin, and fibrillarin).
RESOLFT imaging of live cells expressing vimentin-Kohinoor at low levels of laser power (0.004 J/cm2; less than 1/10,000 ~ 1/375 than the previous RESOLFT).

Potential Applications / Potential Markets:

Applicable for low-phototoxic super-resolution live cell imaging.

State of Development / Opportunity / Seeking:

●Opportunity
・Available for exclusive and non-exclusive licensing
・Exclusive/non-exclusive evaluation for defined period (set up for options)
・Collaborative/supportive research
●Seeking
・Licensing
・Development partner

IP Status:

WO2015/037674 (National phase EP, Issued: JP, US)

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