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.
Moneron
Gael
G
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Hell
Stefan W
SW
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D021621:Imaging, Three-Dimensional; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D015233:Models, Statistical; D016328:NF-kappa B / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D017785:Photons
D005456:Fluorescent Dyes; D016328:NF-kappa B