Far-red organic fluorophores contain a fluorescent impurity.

Far-red organic fluorophores commonly used in traditional and super-resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near-red emission. This near-red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super-resolution localization microscopy.

[1]  G. C. Rogers,et al.  Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization , 2012, Nature Cell Biology.

[2]  Jerker Widengren,et al.  Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy , 2000 .

[3]  Christophe Zimmer,et al.  Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe , 2011, PloS one.

[4]  B. Baird,et al.  Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components. , 2001, Biophysical journal.

[5]  Travis J Gould,et al.  Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. , 2011, Biophysical journal.

[6]  Michael W. Davidson,et al.  Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes , 2007, Proceedings of the National Academy of Sciences.

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

[8]  Carsten Schultz,et al.  Schnelle, zweifarbige Proteinmarkierung an lebenden Zellen für die hochauflösende Mikroskopie , 2014 .

[9]  J. Lippincott-Schwartz,et al.  Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. , 2010, Journal of the American Chemical Society.

[10]  Yongdeng Zhang,et al.  Rational design of true monomeric and bright photoactivatable fluorescent proteins , 2012, Nature Methods.

[11]  Kristin L. Hazelwood,et al.  A bright and photostable photoconvertible fluorescent protein for fusion tags , 2009, Nature Methods.

[12]  E. Pandzic,et al.  STICCS reveals matrix-dependent adhesion slipping and gripping in migrating cells. , 2012, Biophysical journal.

[13]  David R. Liu,et al.  Photoswitching Mechanism of Cyanine Dyes , 2009, Journal of the American Chemical Society.

[14]  X. Xie,et al.  Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells , 2013, Nature Methods.

[15]  Taekjip Ha,et al.  Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. , 2012, Annual review of physical chemistry.

[16]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[17]  Richard P. Haugland,et al.  Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[18]  Carsten Schultz,et al.  Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy. , 2014, Angewandte Chemie.

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

[20]  Benjamin B. Machta,et al.  Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting , 2011, PloS one.

[21]  Suliana Manley,et al.  Photoactivatable mCherry for high-resolution two-color fluorescence microscopy , 2009, Nature Methods.

[22]  Peter J. Verveer,et al.  Chemically Induced Photoswitching of Fluorescent Probes—A General Concept for Super-Resolution Microscopy , 2011, Molecules.

[23]  D. Lamb,et al.  Tracking image correlation: combining single-particle tracking and image correlation. , 2013, Biophysical journal.

[24]  S. Hess,et al.  Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane. , 2012, Angewandte Chemie.

[25]  S. Hess,et al.  Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy , 2013, Journal of optics.

[26]  Suliana Manley,et al.  Superresolution imaging using single-molecule localization. , 2010, Annual review of physical chemistry.

[27]  James H. Adair,et al.  Photophysics of Cy3-encapsulated calcium phosphate nanoparticles. , 2009, Nano letters.

[28]  A. Egner,et al.  Two-color far-field fluorescence nanoscopy based on photoswitchable emitters , 2007 .

[29]  G. V. Zakharova,et al.  Photoprocesses of thiamonomethinecyanine monomers and dimers , 2001 .

[30]  C. Riener,et al.  Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin. , 2000, Bioconjugate chemistry.

[31]  S. Semrau,et al.  Quantification of biological interactions with particle image cross-correlation spectroscopy (PICCS). , 2011, Biophysical journal.

[32]  J. Spudich,et al.  Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Heilemann,et al.  Carbocyanine dyes as efficient reversible single-molecule optical switch. , 2005, Journal of the American Chemical Society.

[34]  W E Moerner,et al.  Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. , 2013, Nano letters.