Depending on the technique applied super resolution light microscopes can resolve structures about 20 nm to 80 nm apart. In comparison, the best widefield fluorescence microscopes have under ideal conditions a resolution limit around 200 nm. The LMF has a microscope 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. STED depletion is done with a 592 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 40 nm to 50 nm can be achieved. This STED setup is ideally suited for one or two color STED with conventional “blue” and “green” dyes including TFP, YFP, and Alexa 488. In addition, our STED is a high-end confocal microscope suitable for most confocal applications.