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