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Switch on the PC Microscope, Scanner Power and Laser Power buttons and turn the Laser Emission key from Off to On. Wait for the computer to boot up, login to the account and then turn on the depletion lasers. These are on the power supply to the left of the microscope, labelled 775-nm, 660-nm and 592-nm. The switches should always be left in the on position, but the keys will need to be turned one quarter-turn clockwise to the Laser On position.
Software start-up
Double-click the LASX icon on the computer desktop. When the splash screen appears, change the STED button from Off to On. Then click OK. Initialise the stage if you need to use the Tile Scan or Mark & Find modes, but before you do so make sure there is no sample on the microscope and that the condenser is raised high enough so the stage doesn’t hit it as it moves.
Once the software has started up the lasers need activating. Click the Configuration button near the top left of the LASX user interface, then click the Laser Config button on the left-hand side of the Configuration window. Turn on the white light laser (WLL) and the depletion lasers STED 592, STED 660 and STED 775. Turn the three depletion lasers up to 100%. The WLL should automatically be at right power (70%). You only need to switch on the 405 Diode if you want to image DAPI, Hoechst or other blue dyes.
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