Leica SP8 inverse (Irchel) - 1: Start-up

"(1)" Switch ON the fluorescence lamp.

"(2)" Switch on the PC.

Sign-in with your ZMB core credentials.

"(3)" Press the main power switch mounted on the right hand side of the table.

Scanner, lasers and microscope will be switched ON. If not, ''

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

check that the microscope control box is switched on.

Please wait for at least 1 min before continuing with next step.

Start the "LAS X" software.

Make sure "machine.xlhw" is selected for "Configuration", and "DMI8" as "Microscope".

Click "OK".

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

An x/y stage initialization is necessary in order to use the Tilescanning, Mark and Find and the Navigator function.

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.

This is a mandatory step as it avoids possible collision of the objectives and stage 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.

Make sure that the stage insert is correctly inserted and flat.

Here correct and flat.

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

Push the condensor arm of the microscope to the back.

Insert your sample with the coverslip facing down.

Adjust the variable clamping range and moveable brackets to properly fix your sample.

Move your sample above 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".

Bring back condenser arm to its straight position.

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

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

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

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.

Remember you can move (back and forth) the condenser arm for ease of access.

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)).

40x water 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). Mandatory for all immersion objectives.

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