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Stimulated emission depletion (STED) fluorescence nanoscopy is a powerful super-resolution imaging technique based on the confinement of fluorescence emission to the central subregion of an observation volume through de-excitation of fluorophores in the periphery via stimulated emission. Here, we introduce stimulated emission double depletion (STEDD) as a method to selectively remove artificial background intensity. In this approach, a first, conventional STED pulse is followed by a second, delayed Gaussian STED pulse that specifically depletes the central region, thus leaving only background. Thanks to time-resolved detection we can remove this background intensity voxel by voxel by taking the weighted difference of photons collected before and after the second STED pulse. STEDD thus yields background-suppressed super-resolved images as well as STED-based fluorescence correlation spectroscopy data. Furthermore, the proposed method is also beneficial when considering lower-power, less redshifted depletion pulses. Stimulated emission double depletion addresses the issue of background in super-resolution imaging and quantitative microscopy through implementation of a two-pulse sequence in a modified stimulated emission depletion set-up. The measured background intensity is removed from each voxel in the acquired images thanks to time-resolved detection.

Red-emitting fluorescent proteins (RFPs) with fluorescence emission above 600 nm are advantageous for cell and tissue imaging applications for various reasons. Fluorescence from an RFP is well separated from cellular autofluorescence, which is in the green region of the spectrum, and red light is scattered less, which allows thicker specimens to be imaged. Moreover, the phototoxic response of cells is lower for red than blue or green light exposure. We have selected a monomeric variant of the tetrameric RFP eqFP611 from Entacmaea quadricolor, mRuby, as a lead structure and used rational protein design to generate RFP variants with improved photochemical properties.Four point mutations were sufficient to obtain mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. The large Stokes shift of 72 nm allows essentially the entire fluorescence emission to be collected even with 640-nm excitation. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy.