Development live cell superresolution microscopy based on a novel fluorescence switching technology

[Speaker] Shigeyuki Namiki:1
[Co-author] Daisuke Asanuma:1, Kenzo Hirose:1
1:Grad, Sch. Med. Univ. Tokyo, Japan

 Superresolution microscopy has revealed the importance of nano-scale arrangement of proteins for the various cell functions such as synaptic transmission and immune function. In particular, superresolution microscopy by single molecule localization method has high spatial resolution and is promising for research on nano-scale distribution of protein of interest in cells. However, live cell superresolution imaging by the introduction of single molecule localization method is delayed due to insufficient photon number of fluorescence and cytotoxicity of reducing agent used for the switching of fluorescence.
 In this study, we aimed to develop superresolution microscopy based on a single molecule localization method which can analyze nanostructures with time resolution of seconds in living cells. For this purpose, we designed a novel protein tag technology, De-Quenching of Organic Dye Emission (De-QODE) tag technology. As a component of De-QODE technology, we synthesized Quenched Organic Dye Emission (QODE) probe consisting of covalently bonded fluorescent dye and quencher. Switching of fluorescence of QODE probe is designed to be regulated by the binding / dissociation of QODE probe and a single chain antibody (scFv) against-quencher (DeQODE tag). Based on this design, we employed dinitrophenyl moiety (DNP) and silicon rhodamine as a quencher and fluorophore, respectively, and we generated scFv against DNP. As we expected, the De-QODE tag expressed as a fusion protein with the target molecules in the cell became strongly fluorescent by the application of the QODE probe in extracellular medium.
 By using De-QODE tag technology, we tried to perform a superresolution imaging by a single molecule localization method. Several synaptic proteins were able to be expressed as a fusion protein with De-QODE tag in cultured hippocampal neurons. The switching of single molecule fluorescence of QODE probe labeled to synaptic proteins was confirmed in the presence of very low concentration of QODE probe in living neurons. By the reconstruction of single molecule localization data, nano-scale distribution of synaptic proteins was visualized in ~20 second resolution. The above results indicate that De-QODE tag technology is suitable for superresolution imaging in living cells and hence contribute to the elucidation of the molecular mechanism underlying various cell functions.

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