TIRF microscope manual

Power Up

  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

Setting up

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

Mag
NA
DIC
Dry/oil
10x
0.40
no
DRY
20x
0.70
no
DRY
40x
0.75
no
DRY
100x
1.46
YES
OIL

 

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.

Acquire images

duke lmcf leica tirf acquisition settings


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

Laser
Lines (nm)
Colour of fluorophores
Examples of fluorophores
405 Diode
405
Deep blue
DAPI
488 Diode
488
Green
GFP, Alexa 488, FITC, CY2
561 Diode
561
Red
Alexa 568, TRITC, CY3
635 Diode
635
Far-red
Alexa 633,CY5

 

Choose suitable exposure times and EM-Gain for each channel

The image window has these settings -

duke lmcf leica TIRF system image window

Press Single image/Capture image/Start to capture images

Saving 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

Shutdown the system

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