Depending on the technique applied super resolution light microscopes can resolve structures about 20 nm to 130 nm apart. In comparison, the best widefield fluorescence microscopes have under ideal conditions a resolution limit around 200 nm. The LMF runnes microscopes based on stimulated emission depletion (STED). With this technique fluorescence is depleted by the STED laser before being emitted from an excited fluorophore. The STED laser light is arranged like a doughnut. The center of the doughnut is not illuminated by the depletion light but by a second conventional laser for excitation. This arrangement is able to create a small fluorescence spot, which is not limited by diffraction as in classical light microscopes.
Our STED is built on an inverted fully motorized confocal microscope from Leica (SP8) with a hardware based focus drift-correction. The SP8 is configured with four detectors (two PMT’s and two high sensitive HyD’s) and two scanners (conventional and 12 kHz resonant). Six laser lines (405 nm, 458 nm, 488 nm, 514 nm, 555 nm, and 640 nm) and a white light laser (WLL) tunable from 470 nm to 670 nm are available for excitation. Suppression of laser light and improvement of the resolution in the STED mode is achieved by time gating available for the two HyD’s. The SP8/STED 3X achieves resolutions below the diffraction limit in lateral as well as axial directions. STED depletion is done with a 592 nm or a 775 nm laser. For excitation in the STED mode the 405 nm, 458 nm, 488 nm, 515 nm or the WLL can be chosen. Under ideal conditions a resolution of 30 nm to 50 nm (lateral) and 120 nm (axial) can be achieved. This STED setup is ideally suited for two or three color STED with conventional “green” or “red/fare red” dyes including TFP, YFP, Alexa 488, Alexa 594, and ATTO647N. For live cell applications, the SP8/STED 3X is equipped with a 93x glycerol objective with a motorized correction collar. In addition, our STED is a high-end confocal microscope suitable for most confocal applications.
Our 2P STED is from Abberior Instuments and combines the Facility line STED microscope and a unique 2Photon STED microscope. The setups is built on a two-photon microscope with a tuneable dual fs-Laser (680nm - 1300nm) and two non-descanned GaAsP-detectors, two pulsed STED lasers @ 595 nm and 775 nm both equipped with a special light modulator for convenient optimization of the STED donut in xy and z, and the Rescue STED, DyMint, and Minfield modules for efficient light dose reduction. In addition, the setup is featuring an adaptive optics module for further correction of excitation and emission wavelengths to increase imaging depths. The Facility line STED module is equipped with four excitation lasers (405 nm, 485 nm, 561 nm, 640 nm) and four highly sensitive spectral detectors (based on avalanche diod technology). The setup is optimized for imaging with increased resolution much deeper in scattering tissue than conventional STED setups.