Starting up the software
- Double-click the LASX icon on the desktop.
- Wait for the splash screen to appear, switch the STED slider from OFF to ON and click OK.
- It takes a while for the software to connect to the hardware but eventually you will see a message asking whether you want to initialise the motorised stage. You have to initialise the stage if you want to use the Tile Scan or Mark & Find modes to do multi-position imaging, otherwise you can just click No.
If you want to initialise the stage then you must first make sure there is no sample on the microscope and that the condenser is raised so the stage doesn't hit it as it moves.
LASX user interface (UI)
This is a link to an overview image of the LASX user interface as it looks after the software has started up.
Activate the lasers
Activate the lasers using the following procedure.
- Click on the Configuration tab.
- Click the Laser Config button.
- Turn on the lasers and drag the STED 592, STED 660 and STED 775 nm sliders up to 100%. Note that the White Light Laser (WLL) should automatically be at 70%. You don't need the 405 Diode on unless you will be imaging DAPI, Hoechst or other blue dyes.
Activating STED and carrying out a STED alignment
- STED is only possible using the specially optically corrected 100x HC PL APO CS2 oil lens, so you must first rotate this into position in the nose piece by selecting it in the drop-down menu in the UI.
- After the 100x lens is in position you can click the Activate STED button in the top right of the left control screen. The Activate STED button will be greyed out until the 100x lens is in position.
- At this stage you may be prompted to carry out an automatic STED alignment procedure. Even if you are not prompted you should carry out the STED alignment because in my experience the lasers can become misaligned over time and the alignment procedure has fixed that.