Leica SR GSD 3D TIRF (Irchel)

Laser Rack

Microscope body ("DMI6000")

Microscope control box ("CTR box")

Fluorescence lamp

Isolation Table

Computer

The control box is always kept ON to maintain steady temperature of the microscope components.

Switch OFF the CTR box, as lasers must be turned ON BEFORE the CTR box.

At the laser rack:

Turn ON the main power switch.

Turn the laser key for the 405 nm laser to "I" in order to enable "LASER Emission".

Switch ON all other lasers. Do NOT turn the keys yet.

The LEDs ("SHG Ready") light shortly up in green and turn then orange.

Once continuously green, turn laser keys to "LASER ON".

Switch ON the control box.

Switch ON the fluorescence lamp.

Once turned on, the lamp should stay on for at least 30 min.

Switch ON the isolation table.

Press "E" in order to enable isolation (indicated by the red LED "ISOL. ON" and display).

Turn ON the computer.

Sign-in with your ZMB core credentials.

Start the "LAS X" software via the desktop icon.

Stop the countdown by clicking on it.

Make sure "DefaultDynamicWidefieldTree.xlhw" and "Standard Configuration" is selected.

Click "OK".

Make sure you are in the "Main" module.

Go to "Configuration".

Select "Laser Config".

Switch "ON" the lasers you need.

The "LASER ENABLED" light should turn white.

Go back to "Acquire".

Select the 160x/1.43 objective in the "LAS X" software.

The 160x/1.43 objective is the only suitable objective for GSD imaging.

Double-check if the objective is correctly inserted (clicked in).

The GSD objective is directly connected to the manual stage (SuMo-Stage) in order to reduce drift.

Push the condenser arm to the back.

Apply "Type F" immersion to your sample or onto the objective (do not touch the lens with the applicator).

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

Move your sample over the objective by using the manual stage knobs.

Movement in y-direction: clockwise moves the stage towards the back.

Movement in x-direction: clockwise moves the stage to the right.

Pull back the condenser arm.

An interlocked "Laser Protection Cover" is attached to the condenser arm to protect from the strong laser illumination needed for imaging.

This cover has to be opened for accessing the sample.

For engaging the lasers during imaging the cover has to be closed.

Proper closure is confirmed by a green LED on the back of the microscope.

In the "LAS X" software:

Choose FLUO to enable widefield illumination.

Choose an appropriate filter cube for your fluorophore.

Set the camera exposure time.

Click "Live".

Use the external controller to focus your sample (max. travel range 400 um).

Switch to "TIRF" mode to enable laser illumination.

Click "Live".

Adapt laser power while being in "Live" mode.

After finding the focus and proper laser and exposure settings switch to the "GSD" wizard.

GSD operation will be explained in another ZMB guide.