Notes after first training session
512 x 512 pixels
notch filter recommended
counting mode always
16 bits (set in configuration > hardware)
Line repetition is equivalent to accumulation. (Faster than frame mode which averages.)
This mode may cause saturation at 8 bits which is why 16 bits used for intensity image recording.
under 1 photon per laser pulse - can only be assessed when scanning live
Do not ramp laser power to get close to 1, 0.2 is ok (may need a few more accumulation passes)
photons per pulse
X axis is photn per pulse, keep below 1, a few ok
Y axis is counts
Only in Live scan
graph in lower left is average, not fitted.
Right click on image -- fast FLIM and counts
hover over pixel and look for events in 100 to 200 range.
more typically 20s to 50s -- then accumulate to 200.
Repetitions-- Line for Live
(if want to use normal image, got to 16 bits to prevent saturation)
Check event count
mult by number accumulations needed to get to 100-200 counts
assess decay curve
Right click on curve to see # of photons (1.5 M is a lot)
Fitting good pulse rate / 4
IRF_ use calculated IRD
Intensity threshold at left. On graph, drag background up.
Click fit upper right.
if x^ 2 (chi squared) doesn't drop when increasing # of fits, then not correct # of fits; drop down to highest fit # where changes.
For FRET, mean + amplitude weighted
FLIM image FIT button, use precise fit to get images.
FLIM image fit. Set threshold. Fit. Can drag over each pixel.
at top FREE viewer
events- mean amplitude for FRET
mean tau for FLIM
FLIM at bottom left- save image --> sends image (pretty picture) to project
Save results saves the fits.
May export data to format compatible with ImageJ or other software. See Guide To Exporting Data.
Article shows lifetime in use.
Especially if you are working with multiple fluorescence probe panels, recommend looking at this paper. We do not have the super res part of the instrument, but the basic lifetime info applies.