Leica SP8 Falcon (Irchel) - Part 1: Start-up

Switch on the red button (underneath the table on the left).

Turns on fluorescence lamp and monitors.

On the right side of the table:

Switch ON the "PC/Microscope", "Scanner Power" and "Laser Power" switches.

Turn the "Laser Emission" key to "ON-1".

Sign-in with your ZMB core credentials.

Start the "LAS X" software. Select:

"machine.xlhw" for "Configuration",

and "DMI8" as "Microscope".

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

Use "Resonant" scanner for fast acquisition and/ or live imaging. However, not advised for FLIM!

Click "OK".

Click "Yes" to initialize the x/y stage. Make sure nothing is placed on the stage.

An x/y stage initialization is necessary to use the Navigator function.

Go to "Configuration".

Select "Laser Config".

Switch "ON" the lasers you will need.

When "ON", the WLL should be at 85% by default.

Go back to "Acquire".

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

This step avoids possible collision during placing of inserts and/or samples.

Select the 10x dry objective via the "LAS X" software.

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

Choose the appropriate sample holder:

The depicted stage insert is usually placed at the microscope.

Other holders (eg. 96 well plates) are stored in a box on a shelve behind the microscope.

If necessary, stage inserts can be easily exchanged as they are held by magnets.

Push the condensor arm of the microscope to the back.

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

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.

To exchange a stage insert Push the condensor arm of the microscope to the back.

Remove the stage insert and place the appropriate one.

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").

Turn z-wheels clockwise to move objectives upwards (closer to the sample). Turn z-wheels counter-clockwise to move objectives downwards (away from sample).

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

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 . For room temperature use the 23°C glycerin media. For live cell imaging the 37°C one.

Water objectives : Use fresh double destiled water.

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

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

Focus your sample as described previously.

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).

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

40x water you can adjust for the correct cover glass thickness.

63x glycerol you can adjust for the cover glass thickness of the corresponding temperature. Upper row for 23°C with the indicated 0.17mm.

Lower row for 37°C and indicated 0.17mm.

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.