Two-photon excitation STED microscopy.

We report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with stimulated-emission depletion (STED). We demonstrate an easy-to-implement and promising laser combination based on a short-pulse laser source for two-photon excitation and a continuous-wave (CW) laser source for resolution enhancement. Images of fluorescent nanoparticles and the immunostained transcription regulator NF kappaB in mammalian cell nuclei exhibit resolutions of <50 nm and approximately 70 nm in the focal plane, respectively, corresponding to a 4-5.4-fold improvement over the diffraction barrier.

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

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

[3]  K. Svoboda,et al.  Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience , 2006, Neuron.

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

[5]  Hell,et al.  Continuous wave excitation two‐photon fluorescence microscopy exemplified with the 647‐nm ArKr laser line , 1998, Journal of microscopy.

[6]  Stefan W. Hell,et al.  Supporting Online Material Materials and Methods Figs. S1 to S9 Tables S1 and S2 References Video-rate Far-field Optical Nanoscopy Dissects Synaptic Vesicle Movement , 2022 .

[7]  S. Hell.  Microscopy and its focal switch , 2008, Nature Methods.

[8]  S. Hell,et al.  STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis , 2006, Nature.

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

[10]  S. Hell,et al.  Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy , 2007, Neuroscience.

[11]  K. Fujita.  [Two-photon laser scanning fluorescence microscopy]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[12]  Christian Eggeling,et al.  STED microscopy reveals crystal colour centres with nanometric resolution. , 2009 .

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

[14]  Volker Westphal,et al.  Nanoscale resolution in the focal plane of an optical microscope. , 2005, Physical review letters.

[15]  S. Hell,et al.  2,2′‐Thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy , 2007, Microscopy research and technique.

[16]  S W Hell,et al.  Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation. , 1998, Optics letters.

[17]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[18]  Andreas Schönle,et al.  Resolution scaling in STED microscopy. , 2008, Optics express.

[19]  Christian Eggeling,et al.  Macromolecular-scale resolution in biological fluorescence microscopy. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Alberto Diaspro,et al.  Two-photon fluorescence excitation and related techniques in biological microscopy , 2005, Quarterly Reviews of Biophysics.

[21]  Stefan W Hell,et al.  Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source. , 2008, Optics letters.