{{tag> howto FLIM acquisition pile-up TCSPC}}
~~TOC~~
====== How to Avoid the Pile-up Effect in FLIM Measurements ======
For a definition of the pile-up effect see here: [[glossary:pile-up_effect|Pile-Up Effect]]
To avoid [[glossary:pile-up_effect|pile-
time of the dark background is usually very long (up to 1/2 of the TCSPC window), which makes the colo... ates have been significantly higher than the pile up limit, or in cases, where the repetition rate has... time of the dark background is usually very long (up to 1/2 of the TCSPC window), which makes the colo... ET efficiency around 0 is expected and also shows up in the histogram.
* On the lower right, the
esponse:** The FLCS pattern matching windows pops up.
{{ separation_of_2_species_with_Image_8.png?600 ... Image_9.png }}
**Response:** Another window pops up containing the measurement files from the workspa... _Image_13.png }}
**Response:** A new window pops up containing the measurement files from the workspa... g }}
**Response:** The TCSPC fitting window pops up.
{{ separation_of_2_species_with_Image_28.png?600
ript_Image_17.png }}
**Response:** A window pops up asking for a file name and a place to store the u... your profile there.
**Response:** A window pops up with the message that the software needs to be re... _Image_21.png?600 }}
**Response:** A window pops up with the message that the software needs to be re
}}
**Response:** The FCS Calibration window pops up.
{{ calibrate_the_confocal_volume_for_fcs_using_t... ript_Image_21.png }}
**Response:** A window pops up asking to confirm the calibration values.
* Cli... rmed whenclosing the software. A message will pop up when closing SymPhoTime 64:
{{ calibrate_the_con
sure NV-centers in bulk diamond.
==== System setup ====
*refractive index of diamond is 2.4. th... omparable to figure 9.
//NOTE:// Measurement setup has to be changed (see [[antibunching measurement... asubramaniana, R. Erdmann, F. Jelezkoa, J. Wrachtrup Fluorescent nanodiamonds for FRET-based monitorin
es, provided that the system has had time to warm up (~15min), and neither the repetition rate nor the... found along the light pathway in a microscope set up.
However, in case of cuvette based measurement ... ever, the dye must be pure, otherwise you may end up with measuring (long lifetime) impurity emission.
the afterflash peak is characteristic for the setup.
To get rid of the afterflash peak, it has been... ns).
*//Higher Delta values// will speed up the calculation but result in a coarser correlati
ions_Image_16.png }}
**Response:** A window pops up asking for a file name and a place to store the u... _Image_19.png?339 }}
**Response:** A window pops up with the message that the software needs to be re
time of the dark background is usually very long (up to 1/2 of the TCSPC window), which makes the colo... t regions, unless you know from the biological setup that it will mainly be the same in all e.g. compl
background is usually very long (with an average up to 1/2 of the TCSPC window), which makes the colo... age was acquired are significantly above the Pile Up limit. It is also needed, if the pulse frequency
rdware with count-rates way beyond classical pile up limit.
For the principle of rapidFLIM - recordi... background is usually very long (with an average up to 1/2 of the TCSPC window), which makes the colo
background is usually very long (with an average up to 1/2 of the TCSPC window), which makes the colo... tem with a dead time significantly above the pile-up-limit or when the image has been acquired with a
PicoQuant to check which extra parts for your set-up are needed to measure polarization.
\\
===== S... _Image_23.png?383 }}
**Response:** A window pops up with the message that the software needs to be re
esponse:** The FLCS pattern matching windows pops up.
{{ using_the_flcs_script_for_spectral_crosstalk_... Image_8.png }}
**Response:** Another window pops up containing the measurement files from the workspa