Zeiss LSM 780 FLIM, startup and measurement

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