Leica SP5 II Installation guide

Quick Manual
How to start and shut down Leica SP 5 II HCA

1) First things first. Check that the lid of the gas chamber is not attachedto
the condenser.Even if you plan to use the gas chamber,take the lid off.
2) Turn on the computer.
3) Change the sample holder if you need to change it. Be careful not to drop
the small screws used to fasten the sample holder into the motorized stage.
4) Turn on the microscope (A), the scanner (B), the laser power (C) and turn
the safety key to On-1 position
5) Turn on the fluorescent lamp if you plan to see fluorescent light through
the binoculars. The intensity ofthe light can be adjusted from the round
knob, but this is usually unnecessary

6) Now you canstart the software LAS-AF and insert your sample into
the sample holder. As the start-up routine moves the stage, it is not
recommended to focus into your sample until after the software has
fully started.
7) Choose either machine – with pump or machine – without pump
according to your needs. Usually the pump is ever onlyused with wellplates
when 63x water immersion objective is needed. Start the software from the
OK button.

8) Move the turret as downas it goes by z down button on
the right side of the microscope and check the stage’s Z
displacement. Objective glasses and plates have focus
roughly 3,5mm from bottom and Mattek dishes have
focus about 1mm above that.
9) Once the software has started, chose Configuration/Lasers and turn on all
the lasers you will use. If you are using Argon laser, adjust its power to 20%
from the slider. If you want to save your data in other than 8 bit format
(recommended if you plan to quantify your data), go to
Configuration/Settings and choose the 12 bit option.

10) If you have saved your measurement settings, you can load them
from Load/Save single setting menu. The user defined settings are saved to
your account are last items on the setting list after pre-programmed Leica
settings. Alternatively, sequential settings can be loaded from sequential
settings.
11) Adjust the detection scheme, open the shutters as needed andadjust
the laser powers as needed. Remember to adjust the beam splitter (BS)
according to your needs
Using CO2
1) To use the gas exchanger, you must open the air andCO2valves as shown
below. Also remember to turn on the gas mixer itself. (Brick, the red box).

2) Put a dish into the adapter.
3) Put a adapter with dish on a stage and put gas
chamber on with tubes attached to gas humidifier.
Shutting down
1) Save your data.
2) Check from reservation system when microscope has a next reservation.
3) Go to Configuration/Lasers and shut down any lasers not needed by the
next user within two hours.
4) Close LAS-AF software
5) Remove your sample and carefully clean the objective(s)
6) If no-one has a reservation withintwo hours ofyour reservation,turn off
the PC/Microscope and Scanner Power switches. Disable the lasers by
turningthe keytoOff-0 position. Wait until Argon laser cooler has stopped
before turning Laser Power switch off.
7) Check if the next person after youwithin 24 hours needs themicroscope at
37C and turnon the heating if needed.
8) Shut down computer if no one is coming within 2 hours, otherwise log off
from windows.