Startup

  • Make a Rhodamine 110 slide (droplet) - lifetime is known, diffusion time is known
  • Use high precision 1.5 cover slip
  • Go to FCS (zeiss software), default GFP setting
  • Count Rate is used (CPM, counts per minute)
  • Adjust the correction collar to highest CPM (optimum is between two worse extremes)
  • Start Experiment

  • Go to Picoquant SymphoTime software
  • FCS tab, test, start
  • Select channel - data channel, but see CPM of each one, first!
  • Pico-FCS-measurement (not test), point, 30 seconds
  • TCSPC resolution 128 ps (faster fit...)
  • Start/stop; select right data channel! (in the right screen)

  • We have CW laser (to test); therefore the Histogram (blue line) is just a high plateau
  • Save measurement (eg. called 2%, for laser intensity)
  • Zeiss ZEN software:
  • use pre-set: "485 external laser, external port"
  • info: pulse-rate is 32 MHz

  • Measure dark background, set no laser power for 485
  • Register Counts

  • Now use Erythrosin Dye slide (coverslip 1.5 high precision, droplet) which has a short lifetime, quenched with KI solution (potassium iodide)
  • get IRF
  • 60% laser = OK
  • but can use optical attenuation (big rotary button: stepwise eg 0->1, small button: continuous adjustment)
  • Attenuation 1 = approx. 0.9 µW

  • Laser power meter: Simply switch on, can set 485
  • Measure and mark laser power.

  • Change to scan
  • pre-set Zeiss settings eg. "488 26x265"
  • there, can also pick pre-sets for FLIM
  • Detectors: are still PMT, with gain 600-700

Measurement

  • "start" in symphotime (1)
  • "start" in zeiss software (2)
  • "stop" in symphotime
  • save image, name it, copy data to word file: Picture of cells, Picture of histogram

  • Do that for each acquisition separately
  • info: for a 2-component fit, >1000 photons per pixel are needed
  • ROI data is displayed in histogram, but warning: has to be told to use that ROI for the fit

  • Select experiment
  • Analyze
  • Calculate fast FLIM
  • Fitting model
  • Import the measured IRF (from the set-up step)
  • Initial fit (change # of parameters used, if necessary)
  • Evaluate amps + their error range
  • Evaluate mean/average lifetime
  • "shift" indicates delay between IRF/population of the early lifetimes that were fit




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