Leica SP8 (Balgrist Campus) - 1: Start up

The corresponding microscope control box can be found on the shelf above the microscope.

Switch ON the fluorescence lamp.

Press the main power switch mounted with a magnet on the top of the computer.

check on the control unit that the "Scanner Power" and "Laser Power" are switched on, and the "Laser Emission" key turned to "On-1".

In brief, Turn ON the power switch.

Cube (temperature) and brick (CO2) are already turned on.

Start the "LAS X" software.

Select the appropriate "Configuration".

"machine with ClimateControl.xlhw" if environmental control is applied and needs to be logged via the software. Make sure the needed components have been switched on (see dedicated guide for live cell imaging).

"machine.xlhw" for standard room temperature (RT) measurements. Please note, environmental control can be also applied here (however, here temperature and CO2 will be not logged via software).

Make sure "DMI8" is selected as "Microscope".

Select either "Resonant" (ON) or non-"Resonant" (OFF) scanning mode.

Click "OK".

The indicator light "MOTOR ON/OFF" at the stage (left hand side) must be green continuously.

Go to "Configuration".

Select "Laser Config".

Switch "ON" the lasers you will need.

Go back to "Acquire".

Lower the objective turret by pressing the downwards "Z" button on the right side of the microscope.

You can now toggle between objectives within the software (drop-down menu).

Select the 10x dry objective.

Additional stage inserts can be found in the cupboard.

Push the condenser arm of the microscope to the back and, if necessary install a different stage insert.

Here correct and flat.

Here not inserted correctly (stage not flat and shaky).

Insert your sample with the coverslip facing down.

Move your sample over the objective with the help of the Joystick.

With the little slider knobs on both sides of the lower wheel you can change between fast and slow movement.

Fast movement - pressing the knobs downwards.

Slow movement - pulling the knobs towards you.

On the touch screen at the microscope choose the light path tab.

Click "FLUO" and choose an appropriate "FLUO-Filtercube" e.g. "DAPI".

Open the "IL-Shutter" (if actived the dot is yellow).

Look through the oculars and focus your sample by using:

the focus wheel on the microscope stand,

or the z-wheel on the external controller (Smart Move).

Toggle between "Z FINE" and "Z COARSE" directly on the Smart Move.

To save your current focus position select the "xyz tab" and the "Focusdrive Z" on the touchscreen of the microscope.

Click the "Upper Focus Limit" button.

Press "Set".

If done successful you will see an upper marker line appearing.

Press the "Lower Limit" button in order to move down (for safe change of the objective or the sample).

Remove your sample and toggle within the software to the objective of choice.

Oil objectives: "Type-F" immersion liquid.

"Glycerin" objectives: either "Type-G" immersion liquid (for RT measurements) or "Glycerin" immersion liquid (for measurements at 37°C).

"Water" objectives: ddH2O (always use fresh).

Mount your sample again and press the "Upper Focus Limit" button.

Focus your sample as described previously.

20x IMM (multi-immersion - Oil, Glycerin or Water) needs to be set to the corresponding immersion media ("OIL", "GLYC" or "0.17-W" (with cover glass) or "W-0" (without cover glass)).

63x glycerol you can correct for the cover glass thickness (0.14-0.18 mm). Standard is usually 0.17 mm.

Make sure that the cap of the spring-loaded front lens is released (working position).

Please save your data from now again on your core storage (network path: \\files.core.uzh.ch\).

Imaging settings: you can load settings from already acquired/stored data.