Stimulated emission depletion microscopy is a technique that relies on the depletion of the excited state fluorophores surrounding the objective focal spot in order to significantly narrow the dimensions and increase resolution through point-spread function engineering. The specimen is raster-scanned with two superimposed laser beams, one of which focuses and excites the fluorophore near the maximum wavelength (as in traditional confocal microscopy). A second laser line coinciding with the emission wavelength of the fluorophore (the STED laser) is shaped into a beam with a central zero node and illuminates the specimen at the periphery of the first laser focal spot to depopulate the excited state. Lateral resolution is dramatically improved using this methodology.
Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Optics Letters 19: 780-782 (1994). The original paper proposing the concept of stimulated emission depletion microscopy. The authors describe the principles of a STED fluorescence microscope and discuss theoretical aspects of how the phenomena can be harnessed for superresolution imaging.
Nanoscale resolution in the focal plane of an optical microscope. Physical Review Letters 94: 143903-4 (2005). The first experimental demonstration of dramatically improved lateral resolution using stimulated emission depletion microscopy. The reduced focal spot dimensions are compared with the traditional diffraction-limited point-spread function of a confocal microscope.
Focal spot of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution. Physical Review Letters 88: 163901-4 (2002). In order to achieve superresolution imaging, the authors apply a dual-objective approach to STED microscopy in order to generate lateral resolution of 33 nanometers. Bacterial membranes were imaged at high resolution using focused light at 760 nanometers.
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proceedings of the National Academy of Sciences (USA) 97: 8206-8210 (2000). A landmark paper that describes breaking the diffraction barrier in both dimensions by quenching excited organic molecules at the rim of the focal spot through stimulated emission. The authors report a nearly spherical point-spread function having a diameter between 90 and 110 nanometers.
STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicles exocytosis. Nature 440: 935-939 (2006). A dramatic and fascinating demonstration of superresolution STED microscopy to image 40-nanometer synaptic vesicles in living neurons, demonstrating that cellular structures with dimensions of a few tens of nanometers can be resolved with conventional far-field optics and visible light.
Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320: 246-249 (2008). The first demonstration of near video-rate superresolution imaging with a focal spot size of 62 nanometers. Synaptic vesicles inside axons of cultured neurons were examined with STED microscopy in a limited 2.5 x 1.8 micrometer field of view.
Two-photon excitation STED microscopy. Optics Express 17: 14567-14573 (2009). Report on the adaptation of STED microscopy for applications using two-photon excitation using a short-pulse laser source. Images of fluorescent nanoparticles and fixed cell nuclei were imaged at resolutions between 50 and 70 nanometers in the focal plane.
Nanoscale resolution in GFP-based microscopy. Nature Methods 3: 721-723 (2006). The authors demonstrate imaging of enhanced green fluorescent protein (EGFP) with STED microscopy to obtain a lateral resolution of approximately 70 nanometers. Among the specimens under study were EGFP-labeled viruses and the endoplasmic reticulum of mammalian cells.
STED microscopy with a supercontinuum laser source. Optics Express 16: 9614-9621 (2008). In a novel application, the authors successfully demonstrate stimulated emission depletion microscopy using a single supercontinuum laser source for both the excitation and STED beams. This combination avoids elaborate laser pulses and enables multicolor imaging.
Dual-color STED microscopy at 30-nm focal-plane resolution. Small 4: 1095-1100 (2008). Cultured hippocampal neurons from neonatal rats were stained to target syntaxin 1 and synaptophysin in order to conduct dual-color superresolution STED microscopy. A resolution of 23 to 35 nanometers in the lateral plane was achieved.