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