Leica SP8 Falcon and STED - Live cell imaging

Start heating at least 30 min before you start your experiment. This allows the chamber and the microscope components to heat up and avoids major drift during your experiment.

Gas bottles for the SP8 Falcon and STED can be found on the left at the entrance of the room.

For the SP8 STED only the CO2 bottle is necessary.

For the SP8 Falcon both CO2 and N2 bottles are needed.

Humidifier: Make sure the humidifier column is filled with water. If not, carefully refill ddH2O. Don't overfill!

Use dishes or well plates suited for imaging. Preferably glass bottom.

Make sure your media does not interfere with the imaging - e.g.use media without phenol red.

Switch ON the temperature controller (the cube).

For the SP8 STED: The temperature controller can be found underneath the microscope.

Define the desired temperature using the arrows.

The yellow number (top) indicates the current temperature measured inside the incubation chamber.

You will find boxes containing the necessary accessories on these cupboards.

Here for the SP8 Falcon,

and here for the SP8 STED.

Take the appropriate cover for the CO2 chamber from box.

You will find the frame inside the incubation chamber.

Place the frame on top of the stage.

There are 2 small metal pins on the frame and two opposing holes in the stage in order to hold it in place.

Slide in the glass/plastic lid to cover the frame.

Adjust the condenser height in case it touches the top of the lid.

N2 (only necessary for SP8 Falcon) and CO2 gas bottles (both SP8 Falcon and SP8 STED).

To open the gas bottles:

CO2 - Open the wall mounted distribution valves labeled "SP8 Falcon" or "SP8 STED" as needed.

CO2 and N2 - Gently open the main tap of the gas bottle.

As well as the small knob on the right.

Please DO NOT CHANGE the pressure control tap!

Switch ON the CO2 controller (the brick).

For SP8 STED: The CO2 controller can be found on top of the WLL/STED laser box (left hand side of the microscope system)

Turn the knob to navigate through the menu options. Small arrow next to the option will show where you are.

Press the knob to select the desired option, in this case "Start".

Use the knob to navigate through the options. The upper row gives the readings inside the chamber.

The lower row indicates the defined settings. In this case CO2 is set to 5% and relative humidity (%rh) should be set to 95%.

ONLY for SP8 Falcon: Set O2 to 20% for normoxia (usually it settles at 18.9-19%).

Adjust the flow - 20 l/h maintains the right CO2 concentration inside the chamber.

Wait until the CO2 concentration and temperature stabilize inside the chamber (approx. 30 min).

Once temperature and CO2 have stabilized you can place your sample/cells.

You can now start setting up your experiment!