1 – Move the microscope transmitted light arm at an angle
Moving the arm at an angle is to get access to the slide holder and to allow the xy-stage to initialize while starting-up the system. (Skip this step on the SP8 X SMD).
2 – Switch on the computer/microscope, scanner and lasers
First, the PC/Microscope.
Second, the Scanner Power.
Third, the Laser Power (use the key to activate emission after switching on the Lasers).
3 – Login and Start the the software
Choose a profile (your department)
Double click the LAS AF icon
After some initializing you are asked to select the Configuration and the Microscope.
Select DMI6000 for the Microscope (most of the time already the default setting). Switch the Resonant Scanner On or Off (Default should be set to Off)
Choose “machine +AFC” as configuration (AFC = Adaptive Focus Control). The “machine” and “SimulatorSP8” setting is only for special applications.
After choosing the configuration a question will eventually pop-up if you want to initialize the XY-Stage. If you do not use stitching or mark and find in your experiment you can answer No, otherwise answer Yes. If you answer yes be sure the stage area is free (see item 1).
When the software start-up has been completed, choose the objective you want to use. Then place your sample and move the microscope transmitted light arm straight.
IMM = Oil/Glycerin/Water (the 20x lens has a manual correction ring, this should be default set on oil, but can be changed to glycerin or water, please set back to oil after using it with glycerin or water).
OIL = For slides.
WATER = For live cells.
Clean after use with tissue and some ethanol. Do Not use anything else and do not press or rub!
Be aware that the microscope LCD screen shows all lenses as IMM (Immersion). This means the 40x oil and the 40x water show the same name. This is why the software should be used to select the objectives.
4 – Visual inspection of your sample
For a visual inspection of your sample in fluorescence mode you have to switch on the fluorescence light source.
You can choose from three different fluorescence filter-blocks by pressing A, I3, or TX2 on the front of the microscope.
A is for DAPI, Alexa405, … (Blue)
I3 is for FITC, GFP, Alexa488, … (green)
TX2 is for TRITC, Cy3, Alexa543, .. (red)
5 – Starting the Lasers
Before setting op your experiment you have to start the lasers you need.
First, go to the configuration tab.
Second, select the “Laser Config”
Third, switch on the lasers you need. Put the WLL on 50% (default is 70% but 50% is enough and extends the Laser lifetime).
If you need the Argon laser you have to set the power to 20% for normal imaging or 60% or higher for FRAP.
6 – Setting up scanning
Most default settings are correct for starting an experiment. To speed up imaging you could set the scanning speed to 600 Hz and enable Bidirectional scanning. This results in a 3 times faster scanning without compromising image quality to much.
7 – What to do at the end of a session
If there is someone using the confocal microscope direct after your session then only clean the lenses with a tissue. First remove the oil, do not rub! Second clean with a little alcohol, again do not rub! If you switch off the microscope completely after your experiment be sure to switch off the lasers first, scanner second. And shutdown the computer before switching off the PC/microscope with the green switch.