Time Gated FLIM Checklist

Setting up imaging

  1. Set HRI gain
    • Maximise gain on HRI to reduce excess noise
      • Use ND filters such that you get 2-3 digital counts per photon.
      • Characterise the HRI with a known flux (see Doug’s thesis)
  2. Choose gating strategy
    • Choose ~7 gates, more when sample lifetime is poorly characterised
    • One in rising edge, one at peak and rest distributed logarithmically
    • Fewer, correctly chosen gates maximises lifetime resolution per unit time
    • Example picture
  3. Set gate width
    • Maximise gate width
    • Ensuring last gate does not catch rising edge of next pulse
  4. Set integration time
    • Brightest sample should fill ~¾ of camera dynamic range
  5. Set number of accumulated frames
    • Ensure sufficient photon flux in dimmest ROI
    • Make sure that background recorded in same way.

Before imaging

  1. Check calibration files
    • Library IRF for this HRI with chosen gate width and gain and relay magnification
    • Delay box calibration at the repetition rate of the laser
    • Plate definition file (XPLT)?
  2. Check HRI triggering
    • Using oscilloscope, should be stable pulse to pulse
    • Check triggering, gate settings are consistent

  3. Make sure condenser is optically blocked (they fluorescence in the blue)

  4. Check laser power stability

    • Measure after fibre

    • Always use single mode fibre

    • Coupling efficiency should be 40-50%
      • Check beam profile and fibre end face

    • Check for slow drift, fluctuations on 0.1-1Hz scale in particular

  5. Measure mono-exponential reference dye to obtain t0 and check system
    • Use 25ps gate spacing
    • Fit reference dye before starting imaging. Should have flat residuals
    • Check for mono-exponential decay, correct lifetime

During imaging

  1. Monitor laser power level
  2. Pray to the FLIM gods

After imaging

  1. Repeat reference dye measurement

  2. Measure camera background
    • Record a series of ~100 images with laser shuttered
    • Use same integration time

  3. Acquire images of untransfected cells USIC

  4. Acquire background image of empty dish with imaging media USIC

  5. Check laser power level