Developing Photoactivatable Fluorescent Proteins for Diffraction-Limited and Superresolution Imaging

Abstract

Photoactivatable fluorescent proteins (PAFPs) are molecules that switch to a new fluorescent state in response to specific light activation, and play vital roles in super-resolution imaging. There are three classes of PAFPs: dark-to-bright photoactivators (PAFPs), irreversible photoconverters (PCFPs), and reversible highlighters (RSFP). However, compared to traditional fluorescent proteins (such as GFP or RFP), only limited PAFPs are available for super-resolution microscopy.Previously, we developed several novel PAFPs, mGeos, with various switching rates, photon numbers and brightness1. And based on the crystal structure of green state mEos2, we evolved two truly monomeric and bright RSFPs, mEos3.1 and mEos3.2, with the good photochemical properties including rapid maturation rate, high photon budget and extremely high labeling density2. Here we present new generation of mGeos2 which are true monomeric PAFPs. These novel fluorescent proteins are suitable for both single color and dual color PALM superresolution imaging, and have a broad brand of applications in traditional fluorescence microscopy such as dynamic tracking and pulse chase labeling of proteins.1. Chang H, Zhang M, Ji W, Chen J, Zhang Y, Liu B, Lu J, Zhang J, Xu P, Xu T, A new series of reversibly switchable fluorescent proteins with beneficial properties for various applications, Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4455-60.2. Zhang M, Chang H, Zhang Y, Yu J, Wu L, Ji W, Chen J, Liu B, Lu J, Liu Y, Zhang J, Xu P, Xu T. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods. 2012 May 13;9(7):727-9.