Remote Control  

Feedback Values

  [Related Topics]   

How to use Count Rates

When adjusting scanning velocity (i.e. the line frequency) or the intensity of the excitation light, it is important to monitor the count rate related feedback values. Several issues are of interest:

  1. statistics within a pixel (image) or time bin (time trace)
  2. total measurement time
  3. pile-up and deadtime effects

The relation between single pixel statistcs, count rate and measurement time is of course straightforward: the lower the count rate and the larger the number of photons needed in a single pixel, the longer the acquisition time needs to be. Either the dwell time on a pixel has to be increased or more repetitions have to be recorded.

However, as the intensity varies throughout the image, it is not sufficient to just monitor the average count rate. Especially the bright regions of the image will in most cases be the most prominent source of information. Thus, the number of counts in the brightest pixel is of interest, too.

The "maxcpp" values (displayed as Max Photons in the SymPhoTime user interface) transfer the number of counts in the brightest pixel of the corresponding images. For TCSPC fitting, it is recommended to acquire at least 1000  photons in the brighter regions.

While a large count rate may in most cases be desirable to reduce measurement time, there is a caveat. Due to deadtime effects both of the TCSPC electronics as well as the detector, distortions of the TCSPC decays are to be expected in each pixel that exceeds a count rate of approximately 1% of the pulsed excitation frequency (or SYNC rate).

For a count rate of 1% of the SYNC rate, given that a photon has been detected in an excitation cycle, there is a 1% chance that at least one second photon will hit the detector within the same excitation cycle. As the detector/TCSPC deadtime will prevent a second photon to be detected, this will lead to a loss of 1% of the total intensity. The problem with this loss is that it is biased: It is predominantly encountered at later photon arrival times within the excitation cycle. Consequentially, the apparent lifetimes will be shortened. While these distortions may be still acceptable at 1%, it is recommended that the average count rate as well as the maximum count rate are well below 10% of the SYNC rate.

The "cps" values both for images and time traces are a means to monitor the average count rate, whereas the "_MaxRate_" (time trace) and the "_MaxCountRate_" (image) values show the count rate in the brightest time bin or pixel. However, it should be noted that these count rates may refer to a combination of detection channels. If the detection channels share a common deadtime (e.g. with PicoHarp in combination with a router), above considerations hold for the sum of all detection channels. If the detection channels are independend with regard to their deadtimes (e.g. the independent channels of a HydraHarp), above considerations hold for a single detection channel.

When an image's line frequency is high, especially in a test measurement the "_MaxCountRate_" value is subject to a large noise. As there typically either is a single photon in a pixel or no photon at all, the "_MaxCountRate_" swiches rather between "on" and "off", where the "off" value is zero and the "on" value is one over the pixel dwell time, i.e. a very large value. In this case it is impossible to use the image as a means to adjust the excitation intensity. To overcome this problem, it is recommended to reduce the line frequency for the test measurement, adjust the excitation intensity, then switch back to a high line frequency for the recorded measurement.

Generally, as time resolved measurements require good statistics, fast scanning is only beneficial if one at least acquires at least about 100 counts in the brightest pixels in a single frame. Otherwise, several frames have to be summed up for the analysis. Then, and a smaller line frequency would have been a good alternative, leading to fewer complications with regard to monitoring the results by means of online analyses. As a side effect, smaller line frequencies reduce the number of necessary frames and thus reduce the number of line start and line end markers in the file. For dark samples/ultrafast scanning this effect can lead to differences in files sizes (and thereby loading times) of a factor of ten and more. => If possible, prefer smaller line frequencies.

In a recorded measurement up to four online analyses can be performed simultaneously. When regarding the fluorescence intensity rather as a function over time than a function over the image pixels, the measurement can be monitored by a countrate meter. In this case it is possible to select a time binning that is independent from the dwell time in a single pixel. This can either be used to implement a kind of gliding average as well as to oversample the scanning process in the fast direction. The latter is a good means to monitor the pile-up limit in cases where the pixel width is much larger than the focal width, thus possibly obscuring bright spots within a pixel. In any case, the "_MaxRate_" parameters of a count rate meter analysis may generally be different from the "_MaxCountRate_" parameter of an image analysis, due to differences of the time binning compared to the pixel dwell time.

The "det" + <"1"…"8"> feedback values (as displayed in the topmost region above the tabbed pages (Test, Measurement and Analysis) serve as a quick means to monitor how many detectors are active and receive photons in an individual measurement. The underlying time resolution is not sufficient to assess the absence or presence of pile-up or deadtime effects.

List of Feedback Values (as transmitted by user defined call-back-function)

   

Read the elements in the list below as construction rules. This means: An entry like the following example (as taken from the test mode part)

  "TCSPCHist" + <"1", "2"> + "_MaxCounts_" + <"Sum", "Sync", "Ch1"…"Ch8">

stands in fact for a "cross product" list with 20 different elements, containing:

  "TCSPCHist1_MaxCounts_Sum",
  "TCSPCHist1_MaxCounts_Sync",
  "TCSPCHist1_MaxCounts_Ch1",
  "TCSPCHist1_MaxCounts_Ch2",
  …
  "TCSPCHist1_MaxCounts_Ch8"
  
  "TCSPCHist2_MaxCounts_Sum",
  "TCSPCHist2_MaxCounts_Sync",
  "TCSPCHist2_MaxCounts_Ch1",
  "TCSPCHist2_MaxCounts_Ch2",
  …
  "TCSPCHist2_MaxCounts_Ch8"


This results e.g. in more than 330 individual names in test mode.

The list is orderd by measurement modi, online analyses, measurement items and calculation sources. Whether a named feedback value is sent or not during a certain measurement is depending on the the user's selective online choices.

In fact, it isn't predictable at program design time by no means! If e.g. none of the four slots for online analyses is set to FastFLIM during an image measurement, then none of the named values commencing with "FastFLIM" will be transmitted, but the countrates of the detectors installed will always be sent. So, you have to take care of robustness, if you want to rely on the feedback values for controlling purposes.

Feedbacks in Test-Mode:

   
Only on start: "ServerVersion" Return value type is rftUInt. Byte[3] = Major Version No., Byte[2] = Minor Version No., Byte[0] + 256 * Byte[1] = Word = Build No.
"ResultingLaserRepetitionRate" The laser repetition rate (Hz; rftInt) as set in the SOM module of the Sepia II by SPT64  (only if a Sepia II is connected)
"Lasername" + <"1"…"8"> The names of the currently assessable lasers as given in SPT64  (only if a Sepia II is connected)
Always: "sync" SYNC rate (Hz)
  "det" + <"1"…"8"> Countrate at detector (cps)

  Analysis   Curve Idx   Value   calculated from… Type Description
TCSPCHist:
  "TCSPCHist" + <"1","2"> + "_MaxCounts_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftInt Number of counts in the maximum TCSPC channel of detector 1/2/3 or the sum over all detectors for TCSPC histogram 1/2 (counts)
  "TCSPCHist" + <"1", "2"> + "_SumCounts_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Integral over the TCSPC histogram 1/2 (counts)
  "TCSPCHist" + <"1", "2"> + "_AvrRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate associated with the TCSPC histogram 1/2 (cps)
  "TCSPCHist" + <"1", "2"> + "_FWHM_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Full width half maximum of the decay curve for detector 1/2/3 or the sum over all detectors for TCSPC histogram 1/2
  "TCSPCHist" + <"1", "2"> + "_FastLT_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Fast lifetime for detector 1/2/3 or the sum over all detectors for TCSPC histogram 1/2
TimeTrace: Sent for every active online analysis that calculates one or more time traces:
  "TimeTrace" + <"1", "2"> + "_MaxRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Maximum counts rate over the visible time trace 1/2 (cps)
  "TimeTrace" + <"1", "2"> + "_AvrRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate over the visible time trace 1/2 (cps)
(equals oldstyle) "cps" + <"1", "2">         rftUInt Average count rate over the visible time trace 1/2 (cps)
FCS: Sent for every active FCS online analysis:
  "FCS" + <"_A", "_B"> + "_MaxRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Maximum counts rate for correlation channel A/B (cps)
  "FCS" + <"_A", "_B"> + "_AvrRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate for correlation channel A/B (cps)
(equals oldstyle) "cps" + <"1", "2">         rftUInt Average count rate for correlation channel A/B (cps)
  "FCS" + <"_A", "_B"> + "_MolCount_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Mean number of molecules within the focal volume for correlation channel A/B
  "FCS" + <"_A", "_B", "_AB"> + "_G0_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat G(0) for correlation channel A/B and G(0) for the cross correlation function between A and B
  "FCS" + <"_A", "_B", "_AB"> + "_MolBrightn_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Molecular brightness for correlation channel A/B (cpm)
(equals oldstyle) "cpm" + <"1", "2">         rftFloat Molecular brightness for correlation channel A/B (cpm)
FastFLIM: Sent for every active imaging online analysis:
  "FastFLIM" + <"1", "2"> + "_MaxPhotons_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftInt Current number of photons in the brightest image pixel (counts) for image online analysis 1/2
(equals oldstyle) "maxcpp" + <"1", "2">         rftUInt Current number of photons in the brightest image pixel (counts)
  "FastFLIM" + <"1", "2"> + "_AvgPhotons_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average number of photons per image pixel (counts) for image online analysis 1/2
  "FastFLIM" + <"1", "2"> + "_MaxCountRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftInt Count rate in the brightest image pixel (cps) for image online analysis 1/2
  "FastFLIM" + <"1", "2"> + "_AvgCountRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate (cps) for image online analysis 1/2
(equals oldstyle) "cps" + <"1", "2">         rftUInt Average count rate (cps) for image online analysis 1/2

Feedbacks in Measurement-Mode:

   
Only on start: "ServerVersion" Return value type is rftUInt. Byte[3] = Major Version No., Byte[2] = Minor Version No., Byte[0] + 256 * Byte[1] = Word = Build No.
"ResultingGroupname" The name of the directory, the measured data and results is saved to. Especially useful, since the name templates are substituted.  (only if "Groupname" was given on request)
"ResultingFilename" The name of the rawdata file, the measured data is saved to. Especially useful, since the name templates are substituted.
"ResultingLaserRepetitionRate" The laser repetition rate (Hz; rftInt) as set in the SOM module of the Sepia II by SPT64  (only if a Sepia II is connected)
"Lasername" + <"1"…"8"> The names of the currently assessable lasers as given in SPT64  (only if a Sepia II is connected)
  "sync" SYNC rate (Hz)
Always: "det" + <"1"…"8"> Countrate at detector (cps)

  Analysis   Curve Idx   Value   calculated from… Type Description
TCSPCHist:
  "TCSPCHist" + <"1"…"4"> + "_MaxCounts_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftInt For each active TCSPC curve: Number of counts in the maximum TCSPC channel (counts) in slot <1/2/3/4>
  "TCSPCHist" + <"1"…"4"> + "_SumCounts_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat For each active TCSPC curve: Integral over the TCSPC histogram (counts) in slot <1/2/3/4>
  "TCSPCHist" + <"1"…"4"> + "_FWHM_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat For each active TCSPC curve: Full width half maximum of the decay curve in slot <1/2/3/4>
  "TCSPCHist" + <"1"…"4"> + "_FastLT_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat For each active TCSPC curve: Fast lifetime of the decay curve in slot <1/2/3/4>
TimeTrace:
  "TimeTrace" + <"1"…"4"> + "_MaxRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Max. count rate over the complete time trace (cps) for each active trace in slot <1/2/3/4>
  "TimeTrace" + <"1"…"4"> + "_AvrRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate over the complete time trace (cps) for each active trace in slot <1/2/3/4>
(equals oldstyle) "cps"         + <"1", "2"> rftUInt Average count rate over the complete time trace (if active) 1/2 (cps)
refer also to 1)
FCS:
  "FCS" + <"1"…"4"> + "_MaxRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Maximum counts rate for correlation channel A/B (cps) in slot <1/2/3/4>
  "FCS" + <"1"…"4"> + "_AvrRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate for correlation channel A/B (cps) in slot <1/2/3/4>
(equals oldstyle) "cps"         + <"1", "2"> rftUInt Average count rate for correlation channel A/B (cps) in slot <1/2>
refer also to 1)
  "FCS" + <"1"…"4"> + "_MolCount_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Mean number of molecules within the focal volume for correlation channel A/B in slot <1/2/3/4>
  "FCS" + <"1"…"4"> + "_G0_" + <"Sum", "Sync", "Ch1"…"Ch8"> +
"_x_" + <"Sum", "Sync", "Ch1"…"Ch8">
rftFloat G(0) for correlation channel A/B in slot <1/2/3/4> and G(0) for the cross correlation function between A and B in slot <1/2/3/4>
  "FCS" + <"1"…"4"> + "_MolBrightn_" + <"Sum", "Sync", "Ch1"…"Ch8"> +
"_x_" + <"Sum", "Sync", "Ch1"…"Ch8">
rftFloat Molecular brightness for correlation channel A/B (cpm) (in slot <1/2/3/4>) and Molecular brightness for correlation channel A/B (cpm) in slot <1/2/3/4>
(equals oldstyle) "cpm"         + <"1", "2"> rftFloat Molecular brightness for correlation channel A/B (cpm)
refer also to 1)
FastFLIM:
  "FastFLIM" + <"1"…"4"> + "_MaxPhotons"     rftInt Current number of photons in the brightest image pixel (counts) for image online analysis in slot 1/2/3/4
(equals oldstyle) "maxcpp"             rftUInt Current number of photons in the brightest image pixel (counts)
refer also to 1)
  "FastFLIM" + <"1"…"4"> + "_AvgPhotons"     rftFloat Average number of photons per image pixel (counts) for image online analysis in slot 1/2/3/4
  "FastFLIM" + <"1"…"4"> + "_MaxCountRate"     rftInt Count rate in the brightest image pixel (cps) for image online analysis in slot 1/2/3/4
  "FastFLIM" + <"1"…"4"> + "_AvgCountRate"     rftFloat Average count rate (cps) for image online analysis in slot 1/2/3/4
(equals oldstyle) "cps1"             rftUInt Average count rate (cps)
refer also to 1)
CountRates:
  "CountRates" + <"1"…"4"> + "_MaxRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Max. count rate over the visible time trace (cps) for each active trace in slot <1/2/3/4>
  "CountRates" + <"1"…"4"> + "_AvgRate_" + <"Sum", "Sync", "Ch1"…"Ch8"> rftFloat Average count rate over the visible time trace (cps) for each active trace in slot <1/2/3/4>
(equals oldstyle) "cps"         + <"1", "2"> rftUInt Average count rate over the visible time trace (if active) 1/2 (cps)
refer also to 1)

1)   Consider, that for downward compatibility with SPT32, the upper mentioned old style names are still supported. However, since there may be more than one analysis online, they are no longer unique per feedback frame but may occure more than once in the frame, embedded in their respective context. So, if you rely on the usage of old style names, you've to take care to know their real context by their neighbour's names.


Related Topics: [Top]   
   [Remote Control]    [Server Dialog]    [Remote Interface]    [RIVarEntry]
   [Support]