Development and characterization of FusionRed variants for widefield fluorescence and single-molecular localization microscopy applications

Sheng-Ting Hung^1*

¹Department of Physics, National Sun Yat-sen University, Kaohsiung, Taiwan

* Presenter:Sheng-Ting Hung, email:sth@mail.nsysu.edu.tw

Fluorescent proteins (FPs) have revolutionized biological imaging and sensing. FusionRed is known as a monomeric red FP (RFP) with low cytotoxicity and good fusion properties in mammalian cells, in addition to the RFP advantage of high penetration depth in tissues. However, the low fluorescence brightness of FusionRed greatly hampers its applications. We identified three key mutations to improve the molecular and cellular brightness through directed evolution using self-built multiparameter fluorescence lifetime cytometry, resulting in a 3.4-fold brighter FusionRed variant, FR-MQV. In addition, we also identified two critical mutations controlling the cis-trans isomerization that affects the brightness of FPs, but offers potential applications in single-molecule localization microscopy (SMLM) as reversiblely switchable FPs. We developed a new method using single molecule blinking and ensemble photobleaching experiments bridged with a three-state model to characterize dark state kinetics of the 2.6-fold brighter FusionRed mutant, FR-MQ, at low irradiances that were previously unmeasurable experimental conditions. Our analysis suggested FR-MQ is a potential candidate for SMLM imaging.

Keywords: Fluorescent proteins, Single-molecule localization microscopy, Imaging, Reversiblely switchable fluorescent proteins, Cis-trans isomerization