Remote Control
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Feedback Values
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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:
- statistics within a pixel (image) or time bin (time trace)
- total measurement time
- 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)
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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.
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Feedbacks in Test-Mode:
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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. |
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"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) |
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"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) |
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"det" + <"1"…"8"> |
Countrate at detector (cps) |
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Analysis |
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Curve Idx |
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Value |
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calculated from… |
Type |
Description |
TCSPCHist: |
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"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) |
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"TCSPCHist" |
+ |
<"1", "2"> |
+ |
"_SumCounts_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Integral over the TCSPC histogram 1/2 (counts) |
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"TCSPCHist" |
+ |
<"1", "2"> |
+ |
"_AvrRate_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Average count rate associated with the TCSPC histogram 1/2 (cps) |
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"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 |
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"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: |
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"TimeTrace" |
+ |
<"1", "2"> |
+ |
"_MaxRate_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Maximum counts rate over the visible time trace 1/2 (cps) |
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"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"> |
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rftUInt |
Average count rate over the visible time trace 1/2 (cps) |
FCS: |
Sent for every active FCS online analysis: |
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"FCS" |
+ |
<"_A", "_B"> |
+ |
"_MaxRate_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Maximum counts rate for correlation channel A/B (cps) |
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"FCS" |
+ |
<"_A", "_B"> |
+ |
"_AvrRate_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Average count rate for correlation channel A/B (cps) |
(equals oldstyle) |
"cps" |
+ |
<"1", "2"> |
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rftUInt |
Average count rate for correlation channel A/B (cps) |
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"FCS" |
+ |
<"_A", "_B"> |
+ |
"_MolCount_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Mean number of molecules within the focal volume for correlation channel A/B |
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"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 |
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"FCS" |
+ |
<"_A", "_B", "_AB"> |
+ |
"_MolBrightn_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Molecular brightness for correlation channel A/B (cpm) |
(equals oldstyle) |
"cpm" |
+ |
<"1", "2"> |
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rftFloat |
Molecular brightness for correlation channel A/B (cpm) |
FastFLIM: |
Sent for every active imaging online analysis: |
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"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"> |
|
|
|
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rftUInt |
Current number of photons in the brightest image pixel (counts) |
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"FastFLIM" |
+ |
<"1", "2"> |
+ |
"_AvgPhotons_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Average number of photons per image pixel (counts) for image online analysis 1/2 |
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"FastFLIM" |
+ |
<"1", "2"> |
+ |
"_MaxCountRate_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftInt |
Count rate in the brightest image pixel (cps) for image online analysis 1/2 |
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"FastFLIM" |
+ |
<"1", "2"> |
+ |
"_AvgCountRate_" |
+ |
<"Sum", "Sync", "Ch1"…"Ch8"> |
rftFloat |
Average count rate (cps) for image online analysis 1/2 |
(equals oldstyle) |
"cps" |
+ |
<"1", "2"> |
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rftUInt |
Average count rate (cps) for image online analysis 1/2 |
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Feedbacks in Measurement-Mode:
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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. |
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"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) |
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"ResultingFilename" |
The name of the rawdata file, the measured data is saved to. Especially useful, since the name templates are substituted. |
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"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) |
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"Lasername" + <"1"…"8"> |
The names of the currently assessable lasers as given in SPT64 (only if a Sepia II is connected) |
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"sync" |
SYNC rate (Hz) |
Always: |
"det" + <"1"…"8"> |
Countrate at detector (cps) |
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Analysis |
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Curve Idx |
|
Value |
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calculated from… |
Type |
Description |
TCSPCHist: |
|
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"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> |
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"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.
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