Two-photon excitation and stimulated emission depletion by a single wavelength.

Super-resolved optical microscopy using stimulated emission depletion (STED) is now a mature method for imaging fluorescent samples at scales beyond the diffraction limit. Nevertheless the practical implementation of STED microscopy is complex and costly, especially since it requires laser beams with different wavelengths for excitation and depletion. In this paper, we propose using a single wavelength to induce both processes. We studied stimulated emission depletion of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) dye with a laser delivering a single wavelength in the near infrared. Fluorescence was excited by two photon absorption with a femtosecond pulse, then depleted by one photon stimulated emission with a stretched pulse. Time-resolved fluorescence decay measurements were performed to determine the depletion efficiency and to prove that fluorescence quenching is not affected by side effects. Numerical simulations show that this method can be applied to super-resolved microscopy.

[1]  A. J. Bain,et al.  Time-resolved stimulated emission depletion in two-photon excited states. , 2003, Biochemical Society transactions.

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

[3]  K. Chou,et al.  Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging. , 2009, Biophysical journal.

[4]  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.

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

[6]  S. Hell.  Increasing the Resolution of Far-Field Fluorescence Light Microscopy by Point-Spread-Function Engineering , 2002 .

[7]  J. Lakowicz,et al.  Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation , 1993, Journal of Fluorescence.

[8]  M. Neil,et al.  Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging. , 2008, Optics letters.

[9]  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.

[10]  D. Camaioni,et al.  Photophysics and cis-trans isomerization of DCM , 1985 .

[11]  Stefan W. Hell,et al.  Fluorescence depletion mechanisms in super-resolving STED microscopy , 2007 .

[12]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[13]  Stefan W. Hell,et al.  Laser-diode-stimulated emission depletion microscopy , 2003 .

[14]  P. R. Hammond,et al.  Laser dye DCM, its spectral properties, synthesis and comparison with other dyes in the red , 1979 .

[15]  Gael Moneron,et al.  Two-photon excitation STED microscopy. , 2009, Optics express.

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

[17]  I Gryczynski,et al.  Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays. , 1994, Biophysical journal.

[18]  T. Klar,et al.  Ultrafast dynamics microscopy , 2000 .

[19]  S. Hell,et al.  STED microscopy with continuous wave beams , 2007, Nature Methods.

[20]  Bernardo L. Sabatini,et al.  Supraresolution Imaging in Brain Slices using Stimulated-Emission Depletion Two-Photon Laser Scanning Microscopy , 2009, Neuron.

[21]  W. Webb,et al.  Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm , 1996 .

[22]  D. Camaioni,et al.  Photoproperties of DCM , 1984 .

[23]  S. Hell,et al.  STED microscopy with a supercontinuum laser source. , 2008, Optics express.

[24]  S. Hell,et al.  Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit , 1995 .

[25]  S. Hell,et al.  Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins. , 2010, Biophysical journal.

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

[27]  Bin Wu,et al.  Monomeric red fluorescent proteins with a large Stokes shift , 2010, Proceedings of the National Academy of Sciences.

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

[29]  A. J. Bain,et al.  Stimulated emission depletion of two-photon excited states , 2002 .

[30]  A. Xia,et al.  Ultrafast excited states relaxation dynamics in solution investigated by stimulated emission from a styryl dye , 2007 .

[31]  R. Heintzmann,et al.  Saturated patterned excitation microscopy--a concept for optical resolution improvement. , 2002, Journal of the Optical Society of America. A, Optics, image science, and vision.

[32]  T. Bonhoeffer,et al.  Live-cell imaging of dendritic spines by STED microscopy , 2008, Proceedings of the National Academy of Sciences.