Background suppression in fluorescence nanoscopy with stimulated emission double depletion

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.

[1]  S. Hell,et al.  Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS). , 2012, Optics express.

[2]  Frederik Görlitz,et al.  STED nanoscopy with fluorescent quantum dots , 2015, Nature Communications.

[3]  R. Medda,et al.  FCS in STED microscopy: studying the nanoscale of lipid membrane dynamics. , 2013, Methods in enzymology.

[4]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[5]  Thomas Dertinger,et al.  Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. , 2007, Chemphyschem : a European journal of chemical physics and physical chemistry.

[6]  G Ulrich Nienhaus,et al.  Confocal optics microscopy for biochemical and cellular high-throughput screening. , 2003, Drug discovery today.

[7]  A. Verkman.  Solute and macromolecule diffusion in cellular aqueous compartments. , 2002, Trends in biochemical sciences.

[8]  G Ulrich Nienhaus,et al.  Where Do We Stand with Super-Resolution Optical Microscopy? , 2016, Journal of molecular biology.

[9]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[10]  Christian Eggeling,et al.  Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. , 2005, Physical review letters.

[11]  Alberto Diaspro,et al.  Encoding and decoding spatio-temporal information for super-resolution microscopy , 2015, Nature Communications.

[12]  Christian Eggeling,et al.  Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells , 2014, Nature Communications.

[13]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[14]  R. Blomley,et al.  Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells , 2013, Nature Communications.

[15]  J. Wiedenmann,et al.  RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP‐J , 2011, The EMBO journal.

[16]  Giuseppe Vicidomini,et al.  STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects , 2013, PloS one.

[17]  N. Morrow,et al.  Physical properties of aqueous glycerol solutions , 2012 .

[18]  S W Hell,et al.  Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes. , 2001, Physical review. E, Statistical, nonlinear, and soft matter physics.

[19]  S. Hell,et al.  Direct observation of the nanoscale dynamics of membrane lipids in a living cell , 2009, Nature.

[20]  Y. Ishitsuka,et al.  Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging , 2015, Scientific Reports.

[21]  Wolfgang J Parak,et al.  A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. , 2009, Nature nanotechnology.

[22]  M. Heilemann,et al.  Direct stochastic optical reconstruction microscopy with standard fluorescent probes , 2011, Nature Protocols.

[23]  Alberto Diaspro,et al.  A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy , 2014, Journal of biophotonics.

[24]  S. Hell.  Far-Field Optical Nanoscopy , 2007, Science.

[25]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[26]  Alberto Diaspro,et al.  Frequency dependent detection in a STED microscope using modulated excitation light. , 2013, Optics express.

[27]  G. Nienhaus,et al.  Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens , 2015, Scientific Reports.

[28]  S. Hell,et al.  Sharper low-power STED nanoscopy by time gating , 2011, Nature Methods.

[29]  S.W. HELL,et al.  A compact STED microscope providing 3D nanoscale resolution , 2009, Journal of microscopy.

[30]  S. Hell,et al.  Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Giuseppe Vicidomini,et al.  STED with wavelengths closer to the emission maximum. , 2012, Optics express.