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--- howto:avoid_pile_up_effect_in_flim_measurements [2015/03/26 12:08] – [Measurement of Pile Up Limit] admin
+++ howto:avoid_pile_up_effect_in_flim_measurements [2015/03/26 12:09] – admin
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-===== Measurement of Pile Up Limit =====
+===== Measurement of the Pile Up Limit =====
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   * For detection of the pile up limit the count rate displayed in the Time Trace below the FLIM image is used.
   * A red line indicates the count rate limit which is 10% of the laser repetition rate.
-  * The **"Time Trace Binning"** should be set to 0.01 ms which equals to 10 µs. The "Number of Points" should be set to 100. In this way, the average count rate is much more realistic especially for images with strongly varying pixel intensities. ((IMPORTANT: this feature is only available in the SymPhoTime 64 Version 2.0 that is currently not available for the Leica SP8 SMD. Here, the bin width in the time trace is fixed at 1 msec averaging many pixels. This might not be suited to set the correct average count rate.))
+  * The **"Time Trace Binning"** should be set to 0.01 ms which equals to 10 µs. The "Number of Points" should be set to 100. In this way, the average count rate is much more realistic especially for images with strongly varying pixel intensities. ((This feature is only available in the SymPhoTime 64 Version 2.0 that is currently not available for the Leica SP8 SMD. Here, the bin width in the time trace is fixed at 1 msec averaging many pixels. This might not be suited to set the correct average count rate.))
   * At a pixel dwell time of 10µs each pixel is displayed as one point in the time trace. At a pixel dwell time of 1 µs the average of 10 pixels are displayed as one point in the time trace.
   * During the Test FLIM measurement the laser intensity is set that the time trace is only at rare instances hitting the red line.
+===== Calculation of the count rate per pixel to determine the Pile-Up effect  =====
+For an already acquired FLIM measurement it is possible to determine the count rate per pixel to determine if a pile-up effect is present.
+Procedure:
+  * Calculate the exposure time per pixel: Multiply the number of frames with the pixel dwell time
+  * Determine the number of photons via the data reader (right mouse click) in a very bright pixel within the FastFLIM image
+  * Calculate the count rate for this bright pixel: Divide the number of photons per pixel by the exposure time per pixel