Reduced dyes enhance single-molecule localization density for live superresolution imaging.

Cell-permeable rhodamine dyes are reductively quenched by NaBH4 into a non-fluorescent leuco-rhodamine form. Quenching is reversible, and their fluorescence is recovered when the dyes are oxidized. In living cells, oxidation occurs spontaneously, and can result in up to ten-fold higher densities of single molecule localizations, and more photons per localization as compared with unmodified dyes. These two parameters directly impact the achievable resolution, and we see a significant improvement in the quality of live-cell point-localization super-resolution images taken with reduced dyes. These improvements carry over to increase the density of trajectories for single-molecule tracking experiments.

[1]  P. Gönczy,et al.  Resolution Doubling in 3D-STORM Imaging through Improved Buffers , 2013, PloS one.

[2]  Suliana Manley,et al.  A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. , 2013, Nature chemistry.

[3]  Joshua C Vaughan,et al.  Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy. , 2013, Journal of the American Chemical Society.

[4]  Shu Jia,et al.  Ultra-bright Photoactivatable Fluorophores Created by Reductive Caging , 2012, Nature Methods.

[5]  Chenglong Xia,et al.  Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes , 2012, Proceedings of the National Academy of Sciences.

[6]  S. Manley,et al.  Multicolor single molecule tracking of stochastically active synthetic dyes. , 2012, Nano letters.

[7]  P. Tinnefeld,et al.  Linking single-molecule blinking to chromophore structure and redox potentials. , 2012, Chemphyschem : a European journal of chemical physics and physical chemistry.

[8]  Jacob Piehler,et al.  Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy. , 2012, Nano letters.

[9]  Mark Bates,et al.  Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging , 2011, Nature Methods.

[10]  E. Betzig,et al.  Facile and General Synthesis of Photoactivatable Xanthene Dyes , 2011, Angewandte Chemie.

[11]  X. Zhuang,et al.  Fast three-dimensional super-resolution imaging of live cells , 2011, Nature Methods.

[12]  Mike Heilemann,et al.  Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging , 2011, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[13]  M. Heilemann,et al.  Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy , 2010 .

[14]  Mike Heilemann,et al.  Live-cell super-resolution imaging with trimethoprim conjugates , 2010, Nature Methods.

[15]  Jan Vogelsang,et al.  Make them blink: probes for super-resolution microscopy. , 2010, Chemphyschem : a European journal of chemical physics and physical chemistry.

[16]  N. Murthy,et al.  A significant improvement of the efficacy of radical oxidant probes by the kinetic isotope effect. , 2010, Angewandte Chemie.

[17]  Jan Vogelsang,et al.  Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy , 2009, Proceedings of the National Academy of Sciences.

[18]  N. Murthy,et al.  Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo. , 2009, Angewandte Chemie.

[19]  Jan Vogelsang,et al.  Superresolution microscopy on the basis of engineered dark states. , 2008, Journal of the American Chemical Society.

[20]  Mike Heilemann,et al.  Photoswitching microscopy with standard fluorophores , 2008 .

[21]  E. Betzig,et al.  Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics , 2008, Nature Methods.

[22]  J. Lippincott-Schwartz,et al.  High-density mapping of single-molecule trajectories with photoactivated localization microscopy , 2008, Nature Methods.

[23]  Terrence J Sejnowski,et al.  Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator. , 2007, Nature chemical biology.

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

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

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

[27]  F. Fonnum,et al.  Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. , 2003, Biochemical pharmacology.

[28]  R. Tsien,et al.  Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. , 1989, The Journal of biological chemistry.

[29]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[30]  Sebastian van de Linde,et al.  Live-cell dSTORM with SNAP-tag fusion proteins. , 2011, Nature methods.