Multiphoton TCSPC FLIM Checklist

Before imaging

  1. Read the Becker and Hickl handbook

  2. Check emission filters

    • 700SP clean-up filter commonly used in addition to spectral filter

    • Check for laser bleedthrough

  3. Check triggering stability

    • Correct sync rate?

  4. Check laser power and mode-locking stability

    • Check for slow drift

  5. Adjust slow delay box

    • Position start of decay at around 20% of imaging window

  6. Acquire gold nano-rod IRF

    • See this paper for more details

    • Available from Sigma, product code 716820

    • Use ~1mW excitation power for IRF

    • Acquire background image USIC

  7. Check power levels

    • Check for negligible change in intensity, lifetime over imaging period

    • Photobleaching rate scales non-linearly with power, consider imaging for longer with lower powers

  8. Reduce background light as far as possible

    • Room lights

    • Monitors

  9. Acquire reference dye measurement to test system

    • Fit reference dye before starting imaging

    • Check for mono-exponential decay, correct lifetime

During imaging

  1. Keep peak *pixel* count rates below at least 5% of repetition rate

    • Understand pulse pileup effects

    • Consider image fill factor when monitoring count rate

  2. Monitor count rate for photobleaching

After imaging

  1. Repeat reference dye measurement to check for system drift

  2. Acquire images of untransfected cells USIC

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

  4. Check laser power level