Developing a novel green photoactivatable fluorophore for the use in super-resolution microscopy

Session

Poster Session

Authors

Tina Sarapa (1), Colin Rickman (1)

Affiliations

1. Heriot-Watt university

Keywords

SMLM, fluorophore, photoactivatable fluorophore, PALM, paGFP, fluorophore development. 

Abstract text

Light microscopy is limited in resolution to approximately half the wavelength of light, as defined by Ernst Abbe. Super-resolution microscopy can bypass this limit of resolution by engineering the shape of light that has been emitted from a sample or separating the emission in time and space. SMLM, or single molecule localisation microscopy, includes a set of techniques based on the latter approach. They use fluorescent probes which can be switched on and off as labels for the structures of interest, and while there are a lot of probes already available, the choice in green photoactivatable fluorophores is severely limited. Currently most photoactivatable (PA) fluorophores used in PALM are red in colour and while many other fluorophores have been developed, there is a lack of a reliable photoactivatable green one. The current existing PA green fluorophore, paGFP, is not as bright as the most commonly used red one, PAmCherry, and is also prone to spontaneous activation which is catastrophic for SMLM imaging.

 

This research focuses on characterising, developing and mutating desired qualities into the new green photoactivatable fluorophore. This will be achieved with site-directed mutagenesis and Gibson cloning, and the proteins will be characterised spectroscopically under several conditions to present the full spectroscopical profile of the protein. Furthermore, the research will include super-resolution imaging (specifically PALM, dSTORM and STED) of the novel protein expressed alone or in fusion in bacteria, yeast cells, and mammalian cells. 

 

The protein and its mutants have been expressed and purified using both IMAC and size exclusion chromatography. Furthermore, full spectroscopical profile has been recorded for the proteins – here is when we have discovered a unique ability of the protein to cycle between on/off states without the loss of fluorescence intensity – a quality common for photoswitchable proteins like Dronpa, but unheard of amongst photoactivatable ones. The protein was also expressed in bacteria and PALM imaging was performed. Obtained images show a good fluorophore density, labelling and high image quality. Because the protein is able to restore its fluorescence, it can be imaged for a lot longer than its closest equivalent paGFP resulting in higher quality images, localisation precision and density. The cyclic ability of the protein could mean there is a new class of reversibly photoactivatable fluorophores, which would be ground-breaking for PALM imaging.