Leica SP8 STED alignment

To be performed before each STED experiment

Mount a gold beads slide, and set up focus using the 93x glycerol objective
Load the pre-set acquisition settings for the alignment of one STED depletion laser. They are aligned separately, using separate settings for each depletion laser. Pre-sets are in the list - but do not use the Leica "Expert" ones, but the custom ones further down the list (source: Hänsch, Sebastian )
This will use the PMT detectors in reflection mode, with the notch-filters removed, using the White Light Laser source as channel 1, and the STED depletion laser as channel 2.
Use XZ scanning to find the coverslip reflection at the interface where the gold beads are resting
Adjust motor collar correction of the objective until coverslip reflection has the brightest centre line, and doesn't skew to one side (up or down).
Use XY scanning to find a gold bead with a nicely shaped and visible white-light laser and STED-laser PSF, and zoom into it maximally.
Adjust the alignment (unlock "expert mode" in the laser configuration screen) by moving the STED depletion ring to be centered around the white-light laser PSF as best as possible.
Evaluate in the Z-direction (confirm using XZ scanning). Double check alignment with other beads on the slide.
Mount a reference slide with known structures (eg. nuclear pores), and use it to confirm that the alignment was succesful.
The system is now ready for use in STED experiments.