Ultra-bright Photoactivatable Fluorophores Created by Reductive Caging

Sub–diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, for which the image resolution depends on the number of photons detected per localization. We report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 104−106 photons per localization and allow imaging of fixed samples with resolutions of several nanometers. This strategy can be applied to many fluorophores across the visible spectrum.

[1]  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.

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

[3]  X. Zhuang,et al.  Whole cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution , 2008, Nature Methods.

[4]  S. Hell,et al.  Fluorescence nanoscopy by ground-state depletion and single-molecule return , 2008, Nature Methods.

[5]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Galston,et al.  Riboflavin-Sensitized Photoöxidation of Indoleacetic Acid and Related Compounds. , 1949, Proceedings of the National Academy of Sciences of the United States of America.

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

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

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

[10]  S. McKinney,et al.  Nonblinking and long-lasting single-molecule fluorescence imaging , 2006, Nature Methods.

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

[12]  H. Ewers,et al.  A simple, versatile method for GFP-based super-resolution microscopy via nanobodies , 2012, Nature Methods.

[13]  Mike Heilemann,et al.  A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. , 2008, Angewandte Chemie.

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

[15]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

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

[17]  Steven Chu,et al.  Subnanometre single-molecule localization, registration and distance measurements , 2010, Nature.

[18]  X. Zhuang,et al.  Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells , 2010, Cell.

[19]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

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

[21]  Thorsten Staudt,et al.  Quantum dot blueing and blinking enables fluorescence nanoscopy. , 2011, Nano letters.

[22]  Thorsten Staudt,et al.  Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. , 2011, Nano letters.

[23]  S. Hell,et al.  Subdiffraction resolution in far-field fluorescence microscopy. , 1999, Optics letters.

[24]  Mike Heilemann,et al.  Super-resolution imaging with small organic fluorophores. , 2009, Angewandte Chemie.

[25]  D. Ballou,et al.  The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments. , 2000, Analytical biochemistry.

[26]  Paul R Selvin,et al.  Polarization effect on position accuracy of fluorophore localization. , 2006, Optics express.