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