Leica SP8 upright BL3 (Irchel) - 1: Start-up

Switch ON the fluorescence lamp.

Switch ON the "Scanner Power", "Laser Power", and turn the "Laser Emission" key to "On-1" (control unit underneath the table).

Switch ON the power knob (on the PC table).

The PC and microscope will be switched on.

Sign-in with your ZMB core credentials.

Start the "LAS X" software.

Make sure "machine.xlhw" is selected as "Configuration", and "DM6000" as "Microscope".

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

Click "OK".

Click "Yes" in order to initialize the x/y stage. Please make sure nothing is placed currently on the stage.

Go to "Configuration".

Select "Laser Config".

Switch ON the lasers you will need.

Go back to "Acquire".

Lower the stage by pressing the "LOWER Z" button on the right side of the microscope.

This is a mandatory step as it avoids possible collision of the stage and objective during exchange of inserts and/or samples.

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

Select the 10x dry objective .

In order to facilitate the focusing it is recommended to start with the 10x dry objective.

Insert your sample with the coverslip facing up and fix it with the two springs.

Move your sample under the objective with the help of the external controller "Smart Move".

Movement in y-direction.

Movement in x-direction.

Toggle between coarse movement "XY Fast" and slow movement "XY Precise".

On the touch screen at the microscope stand choose the objective tab.

Make sure the eyepiece is selected as the "Port - Top Port 100%".

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

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

Open the "IL -Shutter" (if activated 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).

Moving sample upwards (towards objectives) turn z-wheels clockwise/away from you. Moving sample downwards (away from objectives) turn z-wheels counter-clockwise/towards you.

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

The storage of the focal plane is helpful in order to find the focus back if the sample or objective will be changed.

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 successfully 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.

Depending on the objective different immersion media will be used. Apply directly on the sample.

Oil objectives: "Type-F" immersion liquid.

"Glycerin" objectives: "Type-G" immersion liquid .

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

Please further consider the additional information in the next step to guaranty proper image acquisition.

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

Focus your sample as described previously.

For some objectives the correction collar has to be adjusted.

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)).

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

Please, DO NOT remove the objectives for adjustment. They can be also accessed on the system.