Leica AM Total Internal Reflection Fluorescence Manual

1. Fluorescence source
2. Lasers (switch and key switch) - all lasers come on
3. Camera controller
4. Microscope controller
5. Computer
6. (if required) Incubator powerstrip
7. (if required) Heating Unit

Start Leica Tirf LAS AF software

• Check configuration should say "Tirf_hamamatsu" (if not click configuration and select)
• OK

Incubation

• Temp should come up to 37C for both heating units (stage top insert and the chamber)
• CO2 should be set to come up to 5%
• Pump speed 2, CO2 reducing valve 7.5
• Press display to toggle between real% and set%
• Heating unit should be set to heating intensity 2, ventilation speed 3

Make sure the bottom of the coverslip and the objective are clean, any dirt can make TIRF illumination uneven.

Focusing on your sample

It is easiest to choose TL-DIC or FLUO from the "contrast method" options in the software, press the eyepiece button and the shutter button to turn the light one.

• Transmitted light: Press the shutter button to turn on the light
• Fluorscence: Select the cube you want, press the shutter button

TIRF is only possible with the 100x objective

TIRF Alignment (very easy)

• Click autoalign
• Make sure you are focused on your cells
• Tilt back the condensor, adjust xyz on smart to focus and center laser spot on ceiling mark
• Put the TL condensor back into position and close all the incubator doors
• Press align and save
• When it says "finished" you can close the box with the X in the top right

If you have any problems with the TIRF alignment check that you are focused on your sample at the coverslip.

Acquisition mode: Select x y z and t as apropriate (ex XYT will take a single XY plane over time)

Make a setting for each channel you want to acquire - choose contrast methods etc, laser power etc

Choose suitable exposure times and EM-Gain for each channel

The image window has these settings -

Press Single image/Capture image/Start to capture images 

• The files are saved in Leica's .LIF format (same as the SP5)
• The experiments tab shows your files (everytime you pressed "Single Image", "Capture" or "Start")
• Right click for options such as rename, delete, export as Tif . . .
• It is best to not have the .LIF files be extremely large >1GB, consider new experiment for each long timelapse
• If exporting to USB memory etc, it is more stable to save to D:/ then MOVE the data over when the program is closed

Record your use of the system using the CoreResearch booking system

If there is somebody next: logoff and clean-up.

If there is nobody next:

Incubation system must be cooled:

• Turn off heating unit ONLY, turn ventilation speed to 7 (the fan stays on to blow air through the unit and cool it)
• wait about 10 min or until the air and unit feel cool
• then you can turn off the power strip (which turns off the temp control and CO2 unit)

Then everything else backwards:

• Computer from start menu
• Microscope
• Camera controller
• Lasers (switch and key switch)
• Fluorescence source

Please leave the incubator doors closed to help keep dust out of the scope