Abstract
The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M−1cm−1, mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.
Highlights
In recent years, super-resolution fluorescence microscopy methods have evolved at a fast pace[1,2,3,4,5], and wide-spread application of these techniques is fostered by the availability of commercial equipment
We created more than 300 variants by elaborate rational engineering to obtain mGarnet, which differs from mRuby by four point mutations, R67K, T158N, F174L and H197R
In mRuby, the anionic green fluorescent protein (GFP) chromophore is in the trans isomeric state, and its delocalized π -electron system is extended by an acylimine group to form a red-shifted chromophore with absorption and emission peaks at 558 and 605 nm, respectively (Supplementary Fig. S2a)
Summary
Super-resolution fluorescence microscopy methods have evolved at a fast pace[1,2,3,4,5], and wide-spread application of these techniques is fostered by the availability of commercial equipment. Far-red emitting FPs are advantageous as markers for live specimens for multiple reasons: In biological samples, (1) absorption, (2) scattering and (3) autofluorescence are greatly reduced in the red part of the visible spectrum. These are crucial advantages, especially when investigating samples more complex than single cell layers. STED microscopes are typically equipped with 633–640 nm excitation and 750–800 nm emission depletion lasers for excitation of a range of robust dyes that are commercially available These far-red wavelengths can be utilized with mGarnet as a marker in live imaging experiments.
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