PicoQuant GmbH HydraHarp 400 Software V. 3.0.0.1
histogram will go to blocks 0..N-1 of the histogram memory where N is the number of channels that were used
in the T3 mode measurement. A TTTR mode file also contains all control panel settings that were active at the
time of the measurement run. After loading a TTTR mode file, you will find the document title reading
“Histogram from...”. If you choose to save such data you will have to give it a new file name ( *.phu). This is
because now a histogram has been formed, and saving it to the same file name would destroy the original
TTTR mode file. However, you may save the previously formed histogram as if it were obtained in normal
interactive mode, to a standard HydraHarp histogram data file (*.phu).
Reloading T3 mode files serves as a quick diagnostic tool only. For T2 mode files such a feature is currently
not available. Further processing or analysis of TTTR mode data must therefore be performed through external
data analysis software. Such software is available from PicoQuant for a wide range of analysis tasks
(SymPhoTime). Further specialized analysis can be performed by dedicated custom software. If you wish to
save the cost for the commercial TTTR analysis software or if you require special analysis algorithms you may
want to program your own analysis software. For development of your own custom programs, please refer to
the demo code provided. Demo source code is contained on your HydraHarp installation disks and will be
installed by the software setup. Also see section 8.2 for the file format specifications. The paragraph below can
only give an outline.
The first part of a TTTR mode file is a header with the basic setup information, similar to that of the other
modes. What follows after the header is a sequence of 32 bit TTTR records. The TTTR records in the file
consist of different pieces of information in groups of bits. These pieces of information must be extracted by a
bit masking / shifting operation. Their specific layout is different for T2 and T3 mode. In both cases, in addition
to extracting the bit fileds, the software must step through the whole file and interpret the overflow records and
correct the overall time axis accordingly. Details on how this can be done should be looked up in the demo
programs that are installed as part of the software distribution.
Note that T2 and T3 mode files cannot be distinguished by their file extension. They all get stored as *.ptu .
However, you can use the PicoQuant File Info shell extension that will be installed by the HydraHarp software
setup to inspect individual header items of a *.ptu or *.phu file. This includes the measurement mode. Just
right-click on the file in Windows explorer and select Properties. Then look at the tab PQ File Info.
5.3.7. TTTR Mode Measurements with Real–Time Correlation
An important application of the HydraHarp and its TTTR mode is Fluorescence Correlation Spectroscopy.
While TTTR data collection and off–line software correlation has been used for quite some time, PCs have only
recently become fast enough to calculate the correlation function “on the fly” while the data is being collected.
This capability can be very useful in setting up and monitoring of FCS experiments. The HydraHarp software
provides such a real–time software correlator for T2 and T3 mode.
Although it collects the same TTTR mode files, the real–time correlator has its own button on
the Toolbar.
The next figure shows the TTTR mode correlator dialog. Its lower section provides the same file handling and
progress indicators elements as in plain TTTR mode. Please refer to section 5.4 for details. The upper section
of the dialog contains the correlator display and various control elements.
The dialog section Cor(Ax,Bx) allows to select individual input channels for the correlation. A and B are the
virtual input channels to be correlated. All input channels that are ticked in columns A resp. B will be used in
the corresponding correlation channel. If your selection is empty, the heading of the section Cor(Ax,Bx) will turn
red to indicate the problem. If you proceed anyway you will get error messages upon start.
The correlator always calculates the autocorrelations of the two virtual channels (AA and BB) as well as the
cross–correlation (AB). There are three tick boxes that select which of these are shown.
The dialog section Accumul. allows to select an update time for the correlation display. Furthermore it allows to
select between repetitive updates (Osc.) and cumulative collection (Int.). If the latter is chosen one can
manually reset the correlator by clicking the Reset button. Note that all these settings affect only the real–time
correlator. The raw TTTR mode data will be collected continuously and completely independent from the
correlator settings.
The Save button under the Accumul. section allows to save the correlator results as they were last shown. The
saved result is an ASCII file with some header information. The format is self–explanatory. Note that the real–
time correlator is primarily a tool for preview during measurement setup and optimization. It does not allow to
correlate data from TTTR files. If you need the correlation over the entire stream of collected data you need to
make sure you run in Integrating mode and do not press reset during the measurement. If you need the
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