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.
At a glance
- Far-field optical nanoscopy. Science 316, 1153–1158 (2007).
- Where do we stand with super-resolution optical microscopy? J. Mol. Biol. 428, 308–322 (2016). &
- Breaking the diffraction resolution limit by stimulated emission: stimulated-emission–depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994). &
- Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006). , &
- Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006). et al.
- Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006). , &
- Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes. Phys. Rev. E 64, 066613 (2001). , &
- Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000). , , , &
- Tetin, S. Y. (ed.) Fluorescence Fluctuation Spectroscopy (FFS), Parts A and B (Methods in Enzymology Series 518 & 519, Academic Press, 2013).
- Sharper low-power STED nanoscopy by time gating. Nat. Methods 8, 571–573 (2011). et al.
- STED nanoscopy with time-gated detection: theoretical and experimental aspects. PLoS ONE 8, e54421 (2013). et al.
- STED with wavelengths closer to the emission maximum. Opt. Express 20, 5225–5236 (2012). , , , &
- Frequency dependent detection in a STED microscope using modulated excitation light. Opt. Express 21, 210–219 (2013). , &
- A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy. J. Biophoton. 7, 376–380 (2014). et al.
- Encoding and decoding spatio-temporal information for super-resolution microscopy. Nat. Commun. 6, 6701 (2015). et al.
- STED nanoscopy with fluorescent quantum dots. Nat. Commun. 6, 7127 (2015). et al.
- A compact STED microscope providing 3D nanoscale resolution. J. Microsc. 236, 35–43 (2009). , , &
- Monomeric garnet, a far-red fluorescent protein for live-cell STED imaging. Sci. Rep. 5, 18006 (2015). et al.
- RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J. EMBO J. 30, 43–56 (2011). et al.
- A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Nat. Nanotech. 4, 577–580 (2009). , , , &
- Confocal optics microscopy for biochemical and cellular high-throughput screening. Drug Discov. Today 8, 1085–1093 (2003). , , , &
- Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009). et al.
- Scanning STED–FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells. Nat. Commun. 5, 5412 (2014). et al.
- FCS in STED microscopy: studying the nanoscale of lipid membrane dynamics. Methods Enzymol. 519, 1–38 (2013). et al.
- Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Phys. Rev. Lett. 94, 178104 (2005). , , &
- Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF–STED–FCS). Opt. Express 20, 5243–5263 (2012). , , , &
- Physical properties of aqueous glycerol solutions. J. Petrol. Sci. Eng. 98–99, 50–60 (2012). , &
- Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. ChemPhysChem 8, 433–443 (2007). et al.
- Solute and macromolecule diffusion in cellular aqueous compartments. Trends Biochem. Sci. 27, 27–33 (2002).
- Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells. Nat. Commun. 4, 2093 (2013). et al.
- Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor–ligand interactions in living specimens. Sci. Rep. 5, 10149 (2015). et al.
- Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 991–1009 (2011). et al.
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