Fluorescent Proteins for Super-Resolution Microscopy

Abstract

Super-resolution fluorescence microscopy is the method of choice to monitor cellular and subcellular biological processes in live cells. Among the different fluorescent labels presently available, fluorescent proteins (FPs) of the GFP family have the key advantage of being genetically encodable. Localization-based super-resolution microscopy approaches require photoactivatable FPs (PA-FPs) that will change their spectral properties upon irradiation with light of a particular wavelength. To be able to distinguish individual, activated fluorophores from the background and to localize them with high precision, a high photon yield in the activated state and a high dynamic range, i.e., the contrast ratio between the fluorescence of the activated (on) and deactivated (off) states, are essential. In stimulated emission depletion (STED) super-resolution microscopy, the sample is raster-scanned by a tightly focused excitation beam followed by a red-shifted, donut-shaped depletion beam. Any FP used for STED must be exquisitely photostable because it has to go through multiple excitation-depletion cycles while the sample is scanned near its location. Moreover, it must be excitable and de-excitable by the lasers that are typically installed in commercial STED microscopes. Therefore, far-red emitting FPs are preferred.We have selected the green-to-red photoconvertible mEosFPthermo and the far-red emitting mGarnet as templates for targeted protein engineering. Considering that FPs are all very similar and share the same scaffold, an obvious strategy was to identify specific amino acid residues that elicit certain properties to one FP and introduce the corresponding amino acid in the other FP variant by using site-directed mutagenesis. As we will show, such simple rational engineering approaches often do not meet with success, which clearly shows that our current understanding of the physics of proteins is far from being complete.