PicoQuant HydraHarp 400 Instruction manual

HydraHarp 400
Picosecond Histogram
Accumulating Real–time Processor
User's Manual and Technical Data
Version .0.0.1
Time–Correlated
Single Photon Counting System
with USB Interface

PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
Disclaimer
PicoQuant GmbH disclaims all warranties with regard to the supplied software and documentation including all
implied warranties of merchantability and fitness for a particular purpose. In no case shall PicoQuant GmbH be
liable for any direct, indirect or consequential damages or any material or immaterial damages whatsoever
resulting from loss of data, time or profits; arising from use, inability to use, or performance of this software and
associated documentation.
License and Copyright Notice
With the HydraHarp 400 product you have purchased a license to use the HydraHarp software. You have not
purchased any other rights to the software itself. The software is protected by copyright and intellectual
property laws. You may not distribute the software to third parties or reverse engineer, decompile or
disassemble the software or part thereof. You may use and modify demo code to create your own software.
Original or modified demo code may be re–distributed, provided that the original disclaimer and copyright notes
are not removed from it. Copyright of this manual and on–line documentation belongs to PicoQuant GmbH. No
parts of it may be reproduced, translated or transferred to third parties without written permission of
PicoQuant GmbH.
HydraHarp is a registered trademark of PicoQuant GmbH. Other products and corporate names appearing in
this manual may or may not be registered trademarks or subject to copyrights of their respective owners.
PicoQuant GmbH claims no rights to any such trademarks. They are used here only for identification or
explanation and to the owner’s benefit, without intent to infringe.
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Table of Contents
1. Introduction...................................................................................................................................................... 5
2. Primer on Time–Correlated Single Photon Counting.......................................................................................6
2.1. Count Rates and Single Photon Statistics...............................................................................................7
2.2. Timing Resolution.................................................................................................................................... 8
2. . Photon Counting Detectors......................................................................................................................9
2. .1. Photomultiplier Tube (PMT).............................................................................................................9
2. .2. Micro Channel Plate PMT (MCP).....................................................................................................9
2. . . Single Photon Avalanche Photo Diode (SPAD)...............................................................................9
2. .4. Other and Novel Photon Detectors................................................................................................10
2.4. Principles Behind the TCSPC Electronics.............................................................................................10
2.5. Further Reading..................................................................................................................................... 14
. Hardware and Software Installation...............................................................................................................15
.1. General Installation Notes – Read This First.........................................................................................15
.2. Software Installation...............................................................................................................................16
. . Device Installation.................................................................................................................................. 17
.4. Installation Troubleshooting...................................................................................................................18
.5. Uninstalling the Software....................................................................................................................... 18
4. Software Overview......................................................................................................................................... 19
4.1. Starting the Program.............................................................................................................................. 19
4.2. The Main Window.................................................................................................................................. 19
4. . The Toolbar........................................................................................................................................... 21
4.4. The Control Panel.................................................................................................................................. 22
4.5. The Axis Panel....................................................................................................................................... 2
4.6. The Trace Mapping Dialog.....................................................................................................................24
4.7. Other Dialogs......................................................................................................................................... 24
5. Specific Measurement Tasks.........................................................................................................................25
5.1. Setting Up the Input Channels...............................................................................................................25
5.2. Setting Up and Running Interactive Measurements...............................................................................29
5. . Time Tagged Mode Measurements....................................................................................................... 0
5. .1. System Requirements................................................................................................................... 0
5. .2. T2 Mode......................................................................................................................................... 0
5. . . T Mode......................................................................................................................................... 1
5. .4. Running a basic TTTR Mode Measurement.................................................................................. 2
5. .5. External Markers............................................................................................................................
5. .6. Using TTTR Mode Data Files........................................................................................................
5. .7. TTTR Mode Measurements with Real–Time Correlation............................................................... 4
5.4. Time–Resolved Excitation and Emission Spectra.................................................................................. 6
6. Controls and Commands Reference..............................................................................................................41
6.1. Main Window......................................................................................................................................... 41
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PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
6.2. Menus.................................................................................................................................................... 4
6.2.1. File Menu.......................................................................................................................................4
6.2.2. Edit Menu.......................................................................................................................................45
6.2. . View Menu..................................................................................................................................... 46
6.2.4. Help Menu.....................................................................................................................................47
6. . Toolbar................................................................................................................................................... 48
6.4. Control Panel......................................................................................................................................... 50
6.4.1. Sync–Input..................................................................................................................................... 50
6.4.2. Inputs 1…4.................................................................................................................................... 50
6.4. . Inputs 5…8.................................................................................................................................... 51
6.4.4. Acquisition..................................................................................................................................... 51
6.5. Axis Panel.............................................................................................................................................. 54
6.5.1. Time Axis Group............................................................................................................................54
6.5.2. Count Axis Group..........................................................................................................................54
6.6. Trace Mapping Dialog............................................................................................................................ 55
6.7. General Settings Dialog.........................................................................................................................55
6.8. About HydraHarp… Dialog....................................................................................................................56
6.9. Title and Comment Editor...................................................................................................................... 56
6.10. Print Preview Dialog.............................................................................................................................56
7. Problems, Tips & Tricks................................................................................................................................. 58
7.1. PC Performance Issues......................................................................................................................... 58
7.2. USB interface......................................................................................................................................... 58
7. . Histogram Artefacts............................................................................................................................... 58
7.4. Warming Up and Calibration..................................................................................................................59
7.5. Custom Programming of the HydraHarp................................................................................................59
7.6. Software Updates.................................................................................................................................. 59
7.7. Support and Bug Reports...................................................................................................................... 59
8. Appendix........................................................................................................................................................ 61
8.1. Warnings................................................................................................................................................ 61
8.2. Data File Formats.................................................................................................................................. 64
8.2.1. Interactive Histogramming Mode File Format................................................................................64
8.2.2. TTTR Mode File Format.................................................................................................................65
8. . Hardware Technical Data......................................................................................................................66
8. .1. Specifications.................................................................................................................................66
8. .2. Connectors....................................................................................................................................68
8. . . Indicators....................................................................................................................................... 69
8.4. Using the Software under Linux.............................................................................................................70
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1. Introduction
While fluorescence spectroscopic investigations are fairly common today, extracting additional temporal
information from molecules via laser induced fluorescence is a relatively new and much more powerful
technique. The temporal analysis can reveal information about the molecule not available from spectral data
alone. This is why lifetime analysis of laser induced fluorescence by means of Time–Correlated Single Photon
Counting (TCSPC) has gained importance over the recent years. The difference in the fluorescence decay
times of appropriate fluorescent dyes provides a powerful discrimination feature to distinguish molecules of
interest from background or other species. This has made the technique very interesting for sensitive analysis,
even down to the single molecule level.
The acquisition of fluorescence decay curves by means of TCSPC provides resolution and sensitivity that
cannot be achieved with other methods. In practice it is done by histogramming arrival times of individual
photons over many excitation and fluorescence cycles. The arrival times recorded in the histogram are relative
times between laser excitation and corresponding fluorescence photon arrival (start / stop times) ideally
resolved down to a few picoseconds. The resulting histogram represents the fluorescence decay. Although
fluorescence lifetime analysis is a main field of application for the HydraHarp, it is in no way restricted to this
task. Other important applications are e.g. Quantum Optics, Quantum Cryptography (QC) Time–Of–Flight
(TOF) and Optical Time Domain Reflectometry (OTDR) as well as any kind of coincidence correlation.
The HydraHarp 400 is a cutting edge TCSPC system with Universal Serial Bus (USB) interface. Its new
modular design provides a flexible number of input channels and allows innovative measurement approaches.
The timing circuits allow high measurement rates up to 12.5 million counts per second (Mcps) and provide a
time resolution of 1 ps. The modern USB interface provides high throughput as well as ‘plug and play’
installation. The input triggers are programmable for a wide range of input signals. All of them have a
programmable Constant Fraction Discriminator (CFD). These specifications qualify the HydraHarp 400 for use
with most common single photon detectors such as Single Photon Avalanche Diodes (SPADs) and
Photomultiplier Tube (PMT) modules (via preamplifier). The best time resolution is obtained by using Micro
Channel Plate PMTs (MCP–PMT) or modern SPAD detectors. The HydraHarp 400 is perfectly matched to
these detectors and the overall Instrument Response Function (IRF) can be as short as 0 ps FWHM.
Similarly, a wide range of excitation sources can be used, e.g. the interchangeable, easy–to–use LDH diode
laser series, as well as mode locked lasers such as a fs Ti:Sapphire laser. This permits lifetime measurements
down to under ten picoseconds with deconvolution, e.g. using the FluoFit Fluorescence Decay Fit Software.
The HydraHarp 400 can operate in various modes to adapt to different measurement needs. The standard
histogram mode performs real–time histogramming in on–board memory. Other modes are available via
software re-configuration. For instance, two different Time–Tagged–Time–Resolved (TTTR) modes allow
recording of each photon event on separate, independent channels, thereby providing unlimited flexibility in
off–line data analysis such as burst detection and time–gated or lifetime weighted Fluorescence Correlation
Spectroscopy (FCS) as well as picosecond coincidence correlation, using the individual photon arrival times.
The HydraHarp 400 is furthermore supported by a variety of accessories such as pre–amplifiers and an
optional hardware and software add–on allowing control of a monochromator from within the HydraHarp
software. The latter supports automated measurement of Time–Resolved Excitaion/Emission Spectra (TRES).
The HydraHarp software runs on all recent Windows PC platforms with Windows versions 7, 8 and 8.1,
including the x64 editions. It provides functions such as the setting of measurement parameters, display of
measurement results, loading and saving of measurement parameters and decay curves. Important
measurement characteristics such as count rate, count maximum and position, histogram width (FWHM) are
displayed continuously. Data can conveniently be exported via the clipboard, e.g. for immediate processing by
the FluoFit Fluorescence Decay Fit Software. An optional programming library (DLL) enables users to write
custom data acquisition programs for the HydraHarp in all modern programming environments.
For details on the Time–Correlated Single Photon Counting method, please read the next section as well as
our TechNote on TCSPC and consult the literature referenced at the end of section 2.4. Experienced users of
the method should be able to work with the HydraHarp straight away. Nevertheless, we recommend carefully
reading of the sections .2 and . on software and hardware installation to avoid damage. Later, the
comprehensive online–help function of the HydraHarp software will probably let the manual gather dust on the
shelf.
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2. Primer on Time–Correlated ingle Photon Counting
In order to make use of a powerful analysis tool such as time–resolved fluorescence spectroscopy, one must
record the time dependent intensity profile of the emitted light. While in principle, one could attempt to record
the time decay profile of the signal from a single excitation / emission cycle, there are practical problems to
prevent such a simple solution in most cases. First of all, the decay to be recorded is very fast. Typical
fluorescence from organic fluorophores lasts only a few hundred picoseconds to some hundred nanoseconds.
In order to recover fluorescence lifetimes as short as e.g. 200 ps, one must be able to resolve the recorded
signal at least to such an extent, that the exponential decay is represented by some tens of sample points in
time. This means the transient recorder required would have to sample at e.g. 10 ps time steps. This is hard to
achieve with ordinary electronic transient recorders of reasonable dynamic range. Secondly, the light available
may be simply too weak to sample an analog time decay. Indeed the signal may consist of just single photons
per excitation / emission. This is typically the case for single molecule experiments or work with minute sample
volumes / concentrations. Then the discrete nature of the signal itself prohibits analog sampling. Even if one
has more than just a single molecule and some reserve to increase the excitation power to obtain more
fluorescence light, there will be limits, e.g. due to collection optic losses, spectral limits of detector sensitivity or
photo–bleaching at higher excitation power. The solution is Time–Correlated Single Photon Counting (TCSPC).
Since with periodic excitation (e.g. from a laser) it is possible to extend the data collection over multiple
excitation/emission cycles, one can reconstruct the single cycle decay profile from single photon events
collected over many cycles.
The TCSPC method is based on the repetitive, precisely timed registration of single photons of e.g. a
fluorescence signal. The reference for the timing is the corresponding excitation pulse. A single photon detector
such as a Photo Multiplier Tube (PMT) or a Single Photon Avalanche Photodiode (SPAD) is used to capture
the fluorescence photons. Provided that the probability of registering more than one photon per cycle is low,
the histogram of photon arrivals per time bin represents the time decay one would have obtained from a single
shot time–resolved analog recording. The precondition of single photon probability can (and must) be met by
attenuating the light level at the sample if necessary. If the single photon probability condition is met, there will
actually be no photons registered in many of the excitation cycles. The diagrams below illustrate how the
histogram is formed over multiple cycles.
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fluorescence photon
fluorescence photon
start-stop-time 1start-stop-time 2
TCSPC
Histogram
laser pulse many cycles do not
produce a photon

PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
The histogram is collected in a block of memory, where one memory cell holds the photon counts for one
corresponding time bin. These time bins are often (historically) referred to as time channels. In practice, the
registration of one photon involves the following steps: first, the time difference between the photon event and
the corresponding excitation pulse must be measured. For this purpose both signals are converted to electrical
signals. For the fluorescence photon this is done via the single photon detector mentioned before. For the
excitation pulse it may be done via another detector if there is no electrical sync signal supplied by the laser.
Obviously, all conversion to electrical pulses must preserve the precise timing of the signals as accurately as
possible. The actual time difference measurement is done by means of fast electronics which provide a digital
timing result. This digital timing result is then used to address the histogram memory so that each possible
timing value corresponds to one memory cell or histogram channel. Finally the addressed histogram cell is
incremented. All steps are carried out by fast electronics so that the processing time required for each photon
event is as short as possible. When sufficient counts have been collected, the histogram memory can be read
out. The histogram data can then be used for display and e.g. fluorescence lifetime calculation. In the following
we will expand on the various steps involved in the method and associated issues of importance.
2.1. Count Rates and ingle Photon tatistics
It was already mentioned that it is necessary to maintain a low probability of registering more than one photon
per cycle. This was to guarantee that the histogram of photon arrivals represents the time decay one would
have obtained from a single shot time–resolved analog recording (The latter contains the information we are
looking for). The reason for this is briefly the following: Due to dead times of detector and electronics for at
least some tens of nanoseconds after a photon event, TCSPC systems are usually designed to register only
one photon per excitation / emission cycle. If now the number of photons occurring in one excitation cycle were
typically >1, the system would very often register the first photon but miss the following one or more. This
would lead to an over–representation of early photons in the histogram, an effect called ‘pile–up’. This leads to
distortions of the fluorescence decay, typically the fluorescence lifetime appearing shorter. It is therefore crucial
to keep the probability of cycles with more than one photon low.
To quantify this demand, one has to set acceptable error limits and apply some mathematical statistics. For
practical purposes one may use the following rule of thumb: In order to maintain single photon statistics, on
average only one in 20 to 100 excitation pulses should generate a count at the detector. In other words: the
average count rate at the detector should be at most 1 % to 5 % of the excitation rate. E.g. with the diode laser
PDL 800–B, pulsed at 80 MHz repetition rate, the average detector count rate should not exceed 4 Mcps. This
leads to another issue: the count rate the system (of both detector and electronics) can handle. Indeed 4 Mcps
may already be stretching the limits of some detectors and usually are beyond the capabilities of older TCSPC
systems. Nevertheless, one wants high count rates, in order to acquire fluorescence decay histograms quickly.
This may be of particular importance where dynamic lifetime changes or fast molecule transitions are to be
studied or where large numbers of lifetime samples must be collected (e.g. in 2D scanning configurations). This
is why high laser rates (such as 40 or 80 MHz from the PDL 800–B) are important. PMTs can safely handle
TCSPC count rates of up to 10 Mcps, standard (passively quenched) SPADs saturate at a few hundred kcps,
actively quenched SPADs may operate up to 5 Mcps but some types suffer resolution degradation when
operated too fast. Old NIM based TCSPC electronics can handle a maximum of 50 to 500 kcps, newer
integrated TCSPC boards may reach peak rates of 5 to 10 Mcps. With the HydraHarp 400, in each channel
average count rates of 6 Mcps and peak rates up to 12.5 Mcps can be collected. It is worth noting that the
photon arrival times are typically random so that there can be bursts of high count rate and periods of low count
rates. Bursts of photons may still exceed the average rate. This should be kept in mind when comparing count
rates considered here and elsewhere. The specifications for TCSPC systems may interpret their maximum
count rates differently in this respect. This is why another parameter, the so called dead–time is also of interest.
This quantity describes the time the system cannot register photons while it is processing a previous photon
event. The term is applicable both to detectors and electronics. Dead–time or insufficient throughput of the
electronics do not usually have a detrimental effect on the decay histogram or, more precisely, the lifetime to be
extracted from the latter, as long as single photon statistics are maintained. However, the photon losses
prolong the acquisition time or deteriorate the signal to noise ratio (SNR) if the acquisition time remains fixed. In
applications where the photon burst density must be evaluated (e.g. for molecule transition detection) dead–
times can be a problem. The HydraHarp 400 has an extremely short dead time of typically less than 80 ns,
imposing the smallest losses possible with instruments of comparable resolution today.
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2.2. Timing Resolution
The most critical component in terms of timing resolution in TCSPC measurements will usually be the detector.
However, as opposed to analog transient recording, the time resolution of TCSPC is not limited by the pulse
response of the detector. Only the timing accuracy of registering a photon determines the resolution. The
timing accuracy is limited by the timing uncertainty that the detector introduces in the conversion from a photon
to an electrical pulse. This timing error or uncertainty can be as much as ten times smaller than the detector's
pulse response. The timing uncertainties are usually quantified by specifying the rms error (standard deviation)
or the Full Width Half Maximum (FWHM) of the timing distribution or instrument response function (IRF). Note
that these two notations are related but not identical. Good, but also very expensive detectors, notably Micro
Channel Plate PMTs, can achieve timing uncertainties as small as 25 ps FWHM. Lower cost PMTs or SPADs
may introduce uncertainties of 50 to 500 ps FWHM.
The second most critical source of IRF broadening in fluorescence lifetime measurements with TCSPC is
usually the excitation source. While many laser sources can provide sufficiently short pulses, it is also
necessary to obtain an electrical timing reference signal (sync) for comparison with the fluorescence photon
signal. The type of sync signal available depends on the excitation source. With gain switched diode lasers
(e.g. PDL 800–B) a low jitter electrical sync signal is readily available. The sync signal used here is typically a
narrow negative pulse of −800 mV into 50 Ω (NIM standard). The sharp falling edge is synchronous with the
laser pulse (< ps rms jitter for the PDL 800–B). With other lasers (e.g. Ti:Sa) a second detector must be used
to derive a sync signal from the optical pulse train. This is commonly done with a fast photo diode (APD or PIN
diode). The light for this reference detector must be derived from the excitation laser beam e.g. by means of a
semi–transparent mirror. The reference detector must be chosen and set up carefully as it also contributes to
the overall timing error.
Another source of timing error is the timing jitter of the electronic components used for TCSPC. This is caused
by the finite rise / fall–time of the electrical signals used for the time measurement. At the trigger point of
comparators, logic gates etc., the amplitude noise (thermal noise, interference etc.) always present in these
signals is transformed to a corresponding timing error (phase noise). However, the contribution of the
electronics to the total timing error is usually small. For the HydraHarp it is typically 10 ps. Finally, it is always a
good idea to keep electrical noise pick-up low. This is why signal leads should be properly shielded coax
cables, and strong sources of electromagnetic interference should be kept away from the TCSPC detector and
electronics.
The contribution of the time spread introduced by the individual components of a TCSPC system to the total
IRF strongly depends on their relative magnitude. Strictly, the overall IRF is the convolution of all component
IRFs. An estimate of the overall IRF width, assuming independent noise sources, can be obtained from the
geometric sum of the individual components as an rms figure according to statistical error propagation laws:
Due to the squares in the sum, the total will be dominated by the largest component. It is therefore of little value
to improve a component that is already relatively good. If e.g. the detector has an IRF width of 200 ps FWHM,
shortening the laser pulse from 50 ps to 40 ps is practically of no effect. However, it is difficult to specify a
general lower limit on the fluorescence lifetime that can be measured by a given TCSPC instrument. In addition
to the instrument response function and noise, factors such as quantum yield, fluorophore concentration, and
decay kinetics will affect the measurement. However, as a limit, one can assume that under favourable
conditions lifetimes down to 1/10 of the IRF width (FWHM) can still be recovered via deconvolution.
A final time–resolution related issue worth noting is the channel width of the TCSPC histogram. As outlined
above, the analog electronic processing of the timing signals (detector, amplifiers, CFD etc.) creates a
continuous distribution around any true time value. In order to form a histogram, at some point the timing
results must be quantized. This is done by an Analog to Digital Converter (ADC) or an integrated Time–to–
Digital Converter (TDC). This quantization introduces another random error, if chosen too coarse. The
quantization step width (i.e. the resolution) must therefore be small compared with the continuous IRF. As a
minimum sampling frequency, from the information theoretical point of view, one would assume the Nyquist
frequency. That is, the signal should be sampled at least at twice the highest frequency contained in it. For
practical purposes one may wish to exceed this limit where possible, but there is usually little benefit in
sampling the histogram at resolutions much higher than 1/10 of the overall IRF width of the analog part of the
system.
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2.3. Photon Counting Detectors
2.3.1. Photomultiplier Tube (PMT)
A PMT consists of a light–sensitive photo cathode that generates electrons when exposed to light. These
electrons are directed onto a charged electrode called dynode. The collision of the electrons with the dynode
produces additional electrons. Since each electron that strikes the dynode causes several electrons to be
emitted, there is a multiplication effect. After further amplification by multiple dynodes, the electrons are
collected at the anode of the PMT and output as a current. The current is directly proportional to the intensity of
light striking the photo cathode. Because of the multiplicative effect of the dynode chain, the PMT is a photo
electron amplifier of high sensitivity and remarkably low noise. The high voltage driving the tube may be varied
to change the sensitivity of the PMT. Current PMTs have a wide dynamic range, i.e. they can also measure
relatively high levels of light. They are furthermore very fast, so that rapid successive events can be reliably
monitored. One photon on the photo–cathode can produce a short output pulse containing millions of
photoelectrons. PMTs can therefore be used as single photon detectors. In photon counting mode, individual
photons that strike the photo cathode of the PMT are registered. Each photon event gives rise to an electrical
pulse at the output. The number of pulses, or counts per second, is proportional to the light impinging upon the
PMT. As the number of photon events increase at higher light levels, it will become difficult to differentiate
between individual pulses and the photon counting detector will become non–linear. This usually occurs at
1..10 Mcps, dependent on the model. Similarly, in TCSPC applications, individual photon pulses may merge
into one as the count rate increases. This leads to pulse pile–up and distortions of the collected histograms.
The timing uncertainty between photon arrival and electrical output (transit time spread) is usually small
enough to permit time–resolved photon counting at a sub–nanosecond scale. In single photon counting mode
the tube is typically operated at a constant high voltage where the PMT is most sensitive.
PMTs usually operate between the blue and red regions of the visible spectrum, with greatest quantum
efficiency in the blue–green region, depending upon photo–cathode materials. Typical quantum efficiencies are
about 25 %. For spectroscopy experiments in the ultraviolet / visible / near infrared region of the spectrum, a
PMT is very well suited.
Because of noise from various sources in the tube, the output of the PMT may contain pulses that are not
related to the light input. These are referred to as dark counts. The detection system can to some extent reject
these spurious pulses by means of electronic discriminator circuitry. This discrimination is based on the
probability that some of the noise generated pulses (those from the dynodes) exhibit lower signal levels than
pulses from a true photon event. Thermal emission from the cathode that undergoes the full amplification
process can usually not be suppressed this way. In this case cooling of the detector is more helpful.
2.3.2. Micro Channel Plate PMT (MCP)
A Micro Channel Plate PMT consists of an array of glass capillaries (5–25 µm inner diameter) that are coated
on the inside with a electron–emissive material. The capillaries are biased at a high voltage. Like in the PMT,
an electron that strikes the inside wall of one of the capillaries creates an avalanche of secondary electrons.
This cascading effect creates a gain of 10 to 106 and produces a current pulse at the output. Due to the narrow
and well defined electron path inside the capillaries, the transit time spread of the output pulses is much
reduced compared to a normal PMT. The timing jitter of MCPs is therefore sufficiently small to perform time –
resolved photon counting on a picosecond scale, usually outperforming PMTs. Good but also expensive MCPs
can achieve timing uncertainties as low as 25 ps. Microchannel plates are in this respect the best match for the
HydraHarp 400 but they are limited in permitted count rate and provide lower sensitivity towards the red end of
the spectrum compared to suitably optimized SPADs.
2.3.3. ingle Photon Avalanche Photo Diode ( PAD)
Avalanche Photo Diodes (APDs) are semiconductor devices, usually restricted to operation in the visible to
infrared part of the spectrum. Generally, APDs may be used for ultra–low light detection (optical powers <
1 pW), and can also be used as photon–counters in the so–called "Geiger" mode (biased slightly above the
breakdown voltage). In the case of the latter, a single photon may trigger an avalanche of about 10 8 carriers. In
this mode the device can be used as a detector for photon counting with very accurate timing of the photon
arrival. In this context they are also referred to as Single Photon Avalanche Photo Diodes (SPAD). Selected
and small devices may achieve timing accuracies down to 50 ps, but small devices are usually hard to align
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and difficult to focus into. Single–photon detection probabilities of up to approximately 50 % are possible. APDs
are often noisier than PMTs, but can have a greater quantum efficiency. Maximum quantum efficiencies
reported are about 70 %. The popular commercial devices from Excelitas, formerly Perkin Elmer (SPCM–AQR)
provide a timing accuracy of ~400 ps and are specified to have a quantum efficiency of 60 %. These modules
are thermoelectrically cooled for low dark count rate and deliver pre–shaped TTL pulses. They are the most
common detectors for applications where NIR sensitivity is important, e.g. single molecule detection. To
achieve the specified timing accuracy, exact focusing into the center of the active area is necessary. More
recent SPAD designs such as the PDM family from Micro Photon Devices have the benefit of much better
timing resolution and robustness, however, at the expense of a lower sensitivity at the red end of the spectrum.
2.3.4. Other and Novel Photon Detectors
The field of photon detectors is still evolving. Recent developments that are beginning to emerge as usable
products include so called silicon PMTs, Hybrid PMTs, superconducting nanowire detectors and APDs with
sufficient gain for single photon detection in analog mode. Each of these detectors have their specific benefits
and shortcomings. Only a very brief overview can be given here.
Silicon PMTs are essentially arrays of SPADs, all coupled to a common output. This has the benefit of creating
a large area detector that can even resolve photon numbers. The drawback is increased dark count rate and
relatively high afterpulsing.
Hybrid PMTs make use of a combination of a PMT front end followed by an APD structure. The benefits are
good timing and virtually zero afterpulsing while the need for very high voltage is a disadvantage. The hybrid
PMT modules of PicoQuant's PMA Hybrid series alleviate this problem by encapsulating the high voltage
supply, the detector, a peltier cooler and even a protective shutter in a compact housing.
Superconducting nanowires (typically made from NbN) can be used to create photon detectors with excellent
timing performance and high sensitivity reaching into the infrared. The shortcomings for practical purposes are
the extreme cooling requirements and the low fill factor of the wire structures, making it difficult to achieve good
collection efficiencies.
Another class of potentially interesting detectors are recently emerging APDs with very high gain. In
combination with an electronic amplifier they have been shown to detect single photons. As opposed to Geiger
mode, this avoids afterpulsing and allows very fast counting rates. The disadvantage is a high dark count rate,
currently way too high for any practical TCSPC application.
2.4. Principles Behind the TC PC Electronics
For introductory purposes it is worth looking at the design of conventional TCSPC systems first. They consist of
the following building blocks:
The CFD is used to extract precise timing information from the electrical detector pulses that may vary in
amplitude. This way the overall system IRF may be tuned to become narrower and some of the random
background signal can be suppressed. The same could not be achieved with a simple threshold detector
(comparator). Especially with PMTs, constant fraction discrimination is very important, because their pulse
amplitudes vary significantly. Particularly pulses originating from random electrons generated at the dynodes of
the PMT can be suppressed because their avalanches had less time to amplify, and their corresponding output
pulses are small.
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The principle of a classical CFD is the comparison of the original detector signal with an amplified, inverted and
delayed version of itself. The signal derived from this comparison changes its polarity exactly when a constant
fraction of the detector pulse height is reached. The zero crossing point of this signal is therefore suitable to
derive a timing signal independent from the amplitude of the input pulse. In practice the comparison is done by
a summation. The timing is done by a subsequent threshold trigger of the sum signal using a settable level, the
so called zero cross trigger. Newer CFD designs achieve the same objective by differentiating the input signal
and triggering on the zero crossing of the differentiated signal. This is the case for the HydraHarp 400. This has
the benefit of adaption to different detector types without a need for changing physical delay lines.
Making the zero cross level adjustable (slightly above zero) allows to adapt to the noise levels in the given
signal, since otherwise an infinitely small signal could trigger the zero cross comparator. Typical CFDs
furthermore permit setting of a discriminator threshold that determines the lower limit the detector pulse
amplitude must pass. This is primarily used to suppress dynode noise from PMTs.
Similar as for the detector signal, the sync signal must be made available to the timing circuitry. Since the sync
pulses are usually of well defined amplitude and shape, a simple settable comparator (level trigger) is often
sufficient to adapt to different sync sources. Nevertheless, it can be valuable to have a CFD also on the sync
channel. A suitably designed CFD is also usable with pre–shaped signals and simply becomes a level trigger in
this case. This is the case also for the HydraHarp 400. Note that the input signals must have sufficiently fast
rise / fall times in the sub-nanosecond to 10 nanosecond range.
The signals from the two input discriminators / triggers are in conventional systems fed to a Time to Amplitude
Converter (TAC). This circuit is essentially a highly linear ramp generator that is started by one signal and
stopped by the other. The result is a voltage proportional to the time difference between the two signals. In
conventional systems the voltage obtained from the TAC is then fed to an Analog to Digital Converter (ADC)
which provides the digital timing value used to address the histogrammer. The ADC must be very fast in order
to keep the dead time of the system short. Furthermore it must guarantee a very good linearity (over the full
range as well as differentially). These are criteria difficult to meet simultaneously, particularly with ADCs of high
resolution (e.g. 12 bits) as is desirable for TCSPC over many histogram channels.
The histogrammer has to increment each histogram memory cell, whose digital address in the histogram
memory it receives from the ADC. This is commonly done by fast digital logic e.g. in the form of Field
Programmable Gate Arrays (FPGA) or a microprocessor. Since the histogram memory must also be available
for data readout, the histogrammer must occasionally stop processing incoming data. This prevents continuous
data collection. Sophisticated TCSPC systems solve this problem by switching between two or more memory
blocks, so that one is always available for incoming data.
While this section so far outlined the typical structure of conventional TCSPC systems, it is worth noting that
the design of the HydraHarp 400 is somewhat different. Today, it is state–of–the–art that the tasks
conventionally performed by TAC and ADC are carried out by a so called Time to Digital Converter (TDC).
These circuits allow not only picosecond timing but can also extend the measurable time span to virtually any
length by means of digital counters. The HydraHarp 400 does not use one such circuit but one for each input
channel and one for the SYNC input. They independently work on each input signal and provide picosecond
arrival times that then can be processed further, with a lot more options than in conventional TCSPC systems.
In the case of classical TCSPC, this processing consists of a subtraction of the two time figures and
histogramming of the differences. This is identical to the classical start–stop measurements of the conventional
TAC approach. The following figure exemplifies this for one detector channel (Start).
The full strength of the HydraHarp design is exploited by collecting the unprocessed independent arrival times
as a continuous data stream for more advanced analysis. Details on such advanced analysis can be found in
the literature. In this case the on–board memory is reconfigured as a large data buffer (FIFO) so that count rate
bursts and irregular data transfer are decoupled. This permits uninterrupted continuous data collection with
high throughput. This mode of operation is called Time–Tagged Time–Resolved (TTTR) mode. Details are
described in section 5. .
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Forward and Reverse tart– top Mode
For simplicity it is most convenient to assume that the time delay measurement is directly causal, i.e. the laser
pulse causes a photon event and one measures the time delay between laser pulse and the subsequent
photon event. However, most conventional TCSPC systems need to give up this logical concept because of the
high repetition rates of the typical excitation lasers: Since the time measurement circuit cannot know in
advance whether there will be a fluorescence photon, it would have to start a time measurement upon each
laser pulse. Considering that conventionally conversion times are in the region of 0. to 2 ms, any excitation
rate in excess of 0.5 to MHz would overrun the time measurement circuits. In fact they would most of the time
be busy with conversions that never complete, because there is no photon event at all in most cycles. By
reversing the start and stop signals in the time measurement, the conversion rates are only as high as the
actual photon rates generated by the fluorescent sample. These are (and must be) only about 1 to 5 % of the
excitation rate and can therefore be handled easily by the TAC / ADC. The consequence of this approach,
however, is that the times measured are not those between laser pulse and corresponding photon event, but
those between photon event and the next laser pulse. This still works (by software data reversing) but is
inconvenient in two ways:
1) Having to reverse the data leads to unpleasant relocation of the data displayed on screen when the
time resolution is changed.
2) Changing between slow and fast excitation sources requires reconnecting to different inputs, possibly
causing trouble when there is no CFD at one of the inputs.
The HydraHarp 400 is revolutionary in this respect, as it allows to work in forward start stop mode, even with
fast lasers. This is facilitated by two design features:
1) The independent operation of the time digitizers, and
2) a programmable divider in front of the sync input.
The latter allows to reduce the input rate so that the period is at least as long as the dead time. Internal logic
determines the sync period and re–calculates the sync signals that were divided out. It must be noted that this
only works with stable sync sources that provide a constant pulse–to–pulse period, but all fast laser sources
known today meet this requirement within an error band of a few picoseconds. Note: for slow sync sources (< 1
MHz) the sync divider must not be used (set to None). Similarly, the divider must not be used for coincidence
correlation measurements etc. since the sync rate has no influence on this kind of measurements. In summary:
The HydraHarp 400 is designed to always work in forward start–stop mode.
Experimental etup for Fluorescence Decay Measurements with TC PC
The figure below shows a typical setup for fluorescence lifetime measurements using one input channel of the
HydraHarp 400.
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The picosecond diode laser (PDL 800–B) is triggered by its internal oscillator (settable at 2.5, 5, 10, 20 and 40
MHz). The driver unit is physically separate from the actual laser head, which is attached via a flexible cable.
This permits to place the small laser head conveniently anywhere in the optical setup. The light pulses of
typically <70 ps FWHM, are directed toward the sample cuvette via appropriate optics. A neutral density filter
can be used to attenuate the light levels if necessary. Upon excitation, the fluorescent sample will emit light at a
longer wavelength than that of the excitation light. The fluorescence light is filtered out from scattered excitation
light by means of an optical cutoff filter (other configurations may utilize a monochromator here). Then it is
directed to the photon detector, again possibly via some appropriate collection optics, e.g. a microscope
objective or a lens.
As a photon detector the H578 PMT from Hamamatsu is very convenient. It only needs a 12 V supply and
permits an instrument response width of <250 ps, allowing lifetime measurements even much shorter than this
via deconvolution. If a higher time resolution is required, the detector of choice is an MCP–PMT. The electrical
signal obtained from the detector (small negative pulses of typically −10 to −50 mV) is fed to the TCSPC
electronics via a preamplifier (e.g. PAM 105 from PicoQuant). This gives pulses of −100 to −500 mV. Cabling is
double shielded 50 Ω coax cable. If the detector is a SPAD module with TTL output (SPCM–AQR from
Excelitas, formerly Perkin Elmer) then an inverter / attenuator (SIA 400) must be inserted. Modern SPAD
devices like the PDM series from MPD directly provide negative timing signals.
The PDL 800–B laser driver readily provides the electric sync signal needed for the photon arrival time
measurement. This signal (a narrow negative pulse) is also fed to the TCSPC electronics via a high quality
50 Ω coax cable. When using the HydraHarp 400 in combination with the PDL 800–B, the sync pulse adapter
LTT 100 (brass cube with SMA connectors) or a 10 dB attenuator should be inserted directly at the sync output
of the laser driver. This reduces crosstalk into the relatively weak detector signals. If the laser does not provide
an electrical sync signal (e.g. Ti:Sa lasers), a sync detector (photo diode) such as the TDA 200 must be used.
The following figure shows TCSPC histograms obtained with this setup. Excitation source was a PDL 800–B
with a 470 nm laser head running at 20 MHz repetition rate. The narrower (blue) curve represents the system
IRF, here dominated by the detector (PMT). The other curve (dark red) is the fluorescence decay from a
solution of Coumarin 6 in ethanol, a fluorescent dye with fairly short fluorescence lifetime (~2.5 ns). The count
rate was adjusted to <1% of the laser rate to prevent pile–up. The plot in logarithmic scale shows the perfect
exponential nature of the decay curve, as one would expect it. Note that this is obtained even without a
deconvolution of the IRF.
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PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
The approximate mono–exponential fluorescence lifetime can be obtained from a simple comparison of two
points in the curve with count rates in the ratio of 1 : 1/e (e.g. 100 000 : 6 788). In this particular experiment
this resulted in a lifetime estimate of 2.55 ns in good agreement with published results for this dye. For a
precise measurement one would perform a numerical exponential fit with IRF deconvolution (typically
implemented as an iterative reconvolution). This would result in slightly shorter lifetimes since the IRF broadens
the decay. Indeed one can measure lifetimes significantly smaller than the IRF with this method. Additionally,
the rms residue from the fit can be used to assess the quality of the fit and thereby the reliability of the lifetime
measurement. The FluoFit decay fit software package from PicoQuant provides this functionality.
2.5. Further Reading
1. O’Connor, D.V.O., Phillips, D.:
Time–correlated Single Photon Counting
Academic Press, London, 1984
2. Lakowicz, J. R.:
Principles of Fluorescence Spectroscopy, rd Edition
Springer, New York, 2006
. Kapusta, P., Wahl, M., Erdmann, R. (Eds.):
Advanced Photon Counting - Applications, Methods, Instrumentation
Springer Series on Fluorescence, Vol. 15, Springer, 2015
ISBN 978- - 19-156 5-4
4. Ortmann U., Wahl M., Kapusta P.:
Time-resolved fluorescence: Novel technical solutions.
Springer Series on Fluorescence, Vol.5, p.259-275 (2008)
5. Koberling F., Kraemer B., Buschmann V., Ruettinger S., Kapusta P., Patting M., Wahl M., Erdmann R.:
Recent advances in photon coincidence measurements for photon antibunching and full correlation
analysis. Proceedings of SPIE, Vol.7185, 71850Q (2009)
6. Wahl M., Rahn H.-J., Röhlicke T., Kell G., Nettels D., Hillger F., Schuler B., Erdmann R.:
Scalable time-correlated photon counting system with multiple independent input channels.
Review of Scientific Instruments, Vol.79, 12 11 (2008)
7. O’Connor, D.V.O., Ware, W.R., Andre, J.C.:
Deconvolution of fluorescence decay curves. A critical comparison of techniques.
J. Phys. Chem. 8 , 1 –1 4 , 1979
8. Patting M., Wahl M., Kapusta P., Erdmann R.:
Dead-time effects in TCSPC data analysis.
Proceedings of SPIE, Vol.658 , 658 07 (2007)
9. http://www.picoquant.com/scientific/references
Bibliography listing all publications with work based on PicoQuant instruments
10. http://www.picoquant.com/products/category/tcspc-and-time-tagging-modules/hydraharp-400-
multichannel-picosecond-event-timer-tcspc-module#papers
Bibliography listing all publications with work based on the HydraHarp 400
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PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
3. Hardware and oftware Installation
3.1. General Installation Notes – Read This First
When handling the HydraHarp 400 system, be sure to avoid electrostatic discharges, especially when
connecting cables. Before connecting any signals, carefully study the maximum ratings given in section 8. .1.
The HydraHarp software version .0.0.1 works with Windows 7, 8 and 8.11. In order to use the HydraHarp
software with the HydraHarp 400 hardware, a device driver must be installed. The HydraHarp 400 is a USB
‘plug and play’ device, this means the necessary resources and drivers are allocated automatically by the
operating system. Windows will automatically recognize when such a device is connected and load the
appropriate driver. The software setup for the HydraHarp 400 installs the driver so that upon connecting the
device it is readily available for the Windows plug&play device management. This requires that the software
setup is performed before connecting the device. Dependent on your version of Windows you may be
prompted to confirm the final driver installation upon connecting the device. Some older Windows versions
verbosely warn about drivers not being validated by Microsoft. You can safely ignore these warnings.
On some Windows versions you may need administrator status to perform the software setup and de–
installation. For the driver installation, it is needed in any case. Installing as Administrator has the benefit that
you can install the software for use by all users on that computer even if they have limited access rights. The
HydraHarp software will maintain individual settings for each user in the Windows registry. When switching
between users with sessions still running in the background you cannot run the HydraHarp software in multiple
sessions at the same time.
The HydraHarp software .0.0.1 is also suitable for 64-bit versions of Windows, where a suitable 64–bit device
driver will be installed. The HydraHarp software as such runs as a 2–bit application on WOW64.
Important Note for oftware Version 2.0 and higher
Software versions 2.0 and higher require a firmware update of the HydraHarp device if it was purchased before
August 2012 and was not upgraded to new firmware since then. The HydraHarp software can perform this
update when it is first started.
The firmware update requirement has consequences that you must observe:
1. Once the update is performed you will no longer be able to use any HydraHarp software prior to version 2.0.
2. Custom software you may have written for file import of 1.x versions will require minor adaptions.
. You will no longer be able to use custom software based on HHLib.dll prior to version 2.0.
4. Custom HHLib-based software you may have written for 1.x versions will require minor adaptions.
5. After the update you may need small CFD adjustments for any known good setup.
6. In case of a power failure or computer crash during the update the device may become inoperational.
7. Reverting to old firmware or repairing a disrupted update requires a return to factory and may incur costs.
Also note: When the firmware update has been performed the device must be switched off an on again in order
to become operational with the new software.
Important Note for Version 3.0 and higher
Software version .0 and higher uses a new file format with the file name extensions *.ptu for TTTR mode
files or *.phu for histogram data files. The idea of this new format is to place individual header data items not
in a strict file position and order but to “tag” the items by name, so that future additions do not harm existing
software. The new file format will therefore be “future proof” in terms of maintenance and compatibility across
new versions. The downside for now is that users who wrote their own data import routines for older formats
will need to adapt them to the new format one more time. This should be quite easy by means of demo code in
various programming languages provided as part of this release. For the first time this also includes a demo for
LabVIEW. For backward compatibility version .0 can still read (but not write) the previous format version 2.0.
1 Trademarks of Microsoft Inc.
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3.2. oftware Installation
Before installing the new software you should (if applicable) back–up all HydraHarp data files you created in
any older HydraHarp directory (previous versions etc.) as well as any critical data on your PC. For a clean start
we also recommend to uninstall any older HydraHarp software.
The HydraHarp 400 setup files are normally provided on a CD supplied with your HydraHarp device. The
installer program file containing the complete distribution is setup.exe. If you received the software package
by download or any other means of electronic distribution, it will probably be packed in a ZIP–File. In this case
you can unzip this file to a temporary hard disk location of your choice and run the setup from there.
In order to use the HydraHarp software with the HydraHarp 400 hardware, a device driver must be installed.
The HydraHarp 400 is a USB ‘plug and play’ device, this means the necessary resources and drivers are
allocated automatically by the operating system. Windows will automatically recognize when such a device is
connected and load the appropriate driver. Note that the software setup for the HydraHarp 400 installs the
driver so that upon connecting the device it is readily available for the Windows plug&play device management.
It is recommended to perform the software setup before connecting the device. Dependent on your version of
Windows you may be prompted to confirm the final driver installation upon connecting the device. Some
windows versions verbosely warn about drivers not being validated by Microsoft. You can safely ignore these
warnings. Starting with HydraHarp software version 2.1 the driver package will appear as a separate software
installation that can be uninstalled like any other software through the standard control panel mechanisms.
On some Windows versions you will need administrator status to perform the software setup. For the driver
installation you need it in any case. You can run the setup program directly from the CD or from the hard disk
location where you unpacked the electronic distribution.
To perform the installation, insert the installation disk in your CD drive. Open the CD location either
from the Windows desktop or via the Windows Explorer. If you unpacked the setup file to a hard disk
location, open that location. Run setup.exe. The setup program will guide you through the
installation process step by step.
When asked for a destination folder for the new software, please accept the default path or select another
according to your program storage policies. This is where the HydraHarp files will be installed. To avoid
confusion, make sure not to specify the path of an older HydraHarp version that you have not uninstalled or
that of any other program on your PC.
The default location is: \Program Files\PicoQuant\HydraHarp400v30.
Setup will also create a dedicated "program folder" for the new HydraHarp 400 software that will later appear in
the Start Menu. You can accept the default folder name or select another according to your own naming
policies. However, you should make sure not to specify the folder name of an older HydraHarp version that you
have not uninstalled nor the dedicated folder of any other program.
The setup program will install the following files in the chosen destination folder:
HydraHarp.exe the main executable program
HydraHarp.chm the HydraHarp compiled HTML–Help file
Manual.pd the manual in Adobe PDF format
Readme.txt a text file with license information
Readme.txt a text file with program information
pquwstub.dll a helper DLL for operation under Linux with Wine
unins000.exe the uninstaller program file
unins000.dat the uninstaller information file
Setup will also create a subdirectory \ iledemo which contains demo source code for access to
HydraHarp data files in various programming languages. Another folder \sampledata will be created with
samples of HydraHarp data files. Other necessary files such as setup information and the device driver will be
installed in the standard places in your Windows directory tree.
Setup will also install a File Info shell extension that you can use 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 P File Info and the tab P File Comment.
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After the installation the HydraHarp software should be available in the Windows Start Menu under Programs |
Pico uant – HydraHarp 400 v3.0 (or the folder name you chose during setup). However, before running the
software, please proceed to the hardware installation step.
3.3. Device Installation
Make sure to avoid electrostatic discharge when handling the HydraHarp system, especially when connecting
cables. Note that PMT detectors operate with high voltage that may discharge through the signal cable. Make
sure such detectors are switched off and fully discharged before connecting them.
Dependent on the model of the HydraHarp device you purchased it has a USB 2.0 or USB .0 interface.
Consult your PC manual as to whether and where it provides high speed USB 2.0 or super speed USB .0
connectors. The latter are typically blue. The minimum requirement is a USB 2.0 connection. The HydraHarp
system will NOT work through USB 1.x. All current PCs should provide at least USB 2.0 connectivity. Most
recent PCs will provide USB .0 out of the box. If you purchased a HydraHarp with USB .0 interface but the
PC has no USB .0 support you can install a USB .0 adaptor card. It is also possible to run a USB .0
HydraHarp device via USB 2.0 but performance will be poor. Use a proper USB .0 connection whenever
possible.
Always use a quality USB cable rated for the chosen USB speed. The cable length must not exceed 5 metres
(~16 ft). For best reliability we recommended to use the provided cable of metres length. Note that the USB
specification does not allow cable extensions other than dedicated active extension cables or hubs. The
HydraHarp device should work flawlessly through suitable hubs. This is also a valid way of extending the
maximum cable length. After a hub, another cable of up to 5 metres is allowed. Note, however, that hubs may
lower the data throughput. For the same reasons it is recommended not to connect other bandwidth
demanding devices to the same hub.
The inputs for the detector / sync signals are SMA connectors located on the front panel of the HydraHarp 400,
labelled IN and SYNC IN. In case of time resolved fluorescence experiments with a pulsed excitation source,
the sync signal must be connected to the SYNC IN input and the detector signal must be connected to an IN
port. If coincidence correlation experiments between two (or more) detector signals are to be carried out, you
need to decide whether you will be using on–board histogramming or TTTR mode. In the case of on–board
histogramming connect one detector to the SYNC input and one or more to the regular IN ports. Coincidence
timing and histogramming will always be with respect to the SYNC input. In TTTR mode it is possible to
determine the relative timing between all inputs but this requires off–line data analysis (see section 5. ).
All inputs are terminated with 50 Ω internally. Note that they need negative going input signals. Use quality
50 Ω coax leads with appropriate connectors. For interfacing to BNC connectors use standard adaptors.
Carefully screw on the SMA connectors for sync and detector until they are moderately tight. Do not use
wrenches. Connect the cable ends to the appropriate signal sources (50 Ω) in your experimental setup. The
HydraHarp 400 inputs accept negative pulses going from 0 V to max. –1 V. Both inputs should be operated
with similar pulse amplitudes to minimize crosstalk. The optimum range is –100 mV to –500 mV. Below this
range you may pick up noise, above there may be crosstalk. Most PMT and MCP detectors will require a pre–
amplifier to reach enough signal level. TTL–SPAD–detectors (e.g. Excelitas SPCM–AQR) must be connected
through a pulse inverter (PicoQuant SIA 400). Weak PMT detectors should be connected through a 20 dB high
speed pre–amplifier. MCP–PMT detectors should be connected through an amplifier with slightly higher gain.
Suitable devices are available from PicoQuant. Be sure to switch the high voltage supply of PMTs off and allow
their electrodes to discharge before connecting them. Their high voltage charge may damage the pre–amplifier.
Observe the allowed maximum ratings for the input signal levels. If you are not sure what signals your setup
delivers, use a fast oscilloscope to check the signal level and shape before connecting them to the HydraHarp.
All signals should have rise/fall times of no more than 2 ns. Slower signals may not be seen by the HydraHarp
and will certainly degrade timing accuracy.
When using the HydraHarp 400 in combination with the PDL 800–B, the sync pulses from the laser driver
should be attenuated by 10 dB to 15 dB to fall in the optimum range for smallest crosstalk.
Do not connect anything other than dedicated hardware to the other HydraHarp 400 connectors. They are
provided for hardware expansion (notably experiment control) and must not be used otherwise. See
section 8. .2 for pin assignments. It is recommended to start instrument setup without anything connected to
the control ports.
After connecting the HydraHarp to the PC via USB for the first time, Windows should detect the device and
perform the final driver installation. Note that the software setup for the HydraHarp 400 pre–installs the driver.
Dependent on your version of Windows you may be prompted to confirm the final driver installation. Some
older windows versions verbosely warn about drivers not being validated by Microsoft. You can safely ignore
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these warnings. After driver installation Windows should report the device as ready to use. If you wish to verify
correct installation you can check the presence of 'HydraHarp 400' as a USB device in the Windows Device
Manager. For troubleshooting see the next section.
3.4. Installation Troubleshooting
After completion of the software setup and device installation the HydraHarp device should be listed in the
Windows Device Manager under the USB tree. Right click My Computer > left click Properties > Device
Manager to check if the device is free of conflicts and / or if the device driver is installed correctly. Under USB
look for a device named HydraHarp 400 and inspect its Properties.
You can also use the Windows system information facilities (Start > Run > msinfo32). In the System
Information utility inspect Software environment > System Drivers to check if the HydraHarp device driver
PQUSB.SYS (PQUSB64.SYS on x64) is correctly installed and running.
You can also repeat the software installation if necessary. Disconnect the device, uninstall the software and
repeat the setup procedure. Make sure the software is not installed in multiple places. If this does not resolve
the problem, try a different computer. If problems persist, see section 7 for support.
3.5. Uninstalling the oftware
Before uninstalling the HydraHarp software you should back–up all valuable data files you might have created
in the HydraHarp installation directory.
Do not manually delete any program files from the installation folder as it will render a clean uninstall
impossible. Also do not delete any driver files manually.
To uninstall the HydraHarp software from your PC you may need administrator rights (dep. on Windows
version and security settings). Go to Control Panel > Add/Remove Software or Programs and select
Pico uant – HydraHarp 400 vx.x for un–installation. This will remove all files that were installed by the
HydraHarp setup program but not the user data that may have been stored. If there was user data in the
program folders or any subfolders, these will not be deleted by the uninstall program. If intended, you need to
delete these files or folders manually. Nevertheless, it is recommended to back–up valuable measurement data
before uninstalling the software.
Note that un–installation of the data acquisition software does not uninstall the device driver. Do not delete the
driver software from within Device Manager. Starting with HydraHarp software version 2.1 the driver package
appears as a separate software installation that can be uninstalled like any other software through the standard
Windows Control Panel mechanisms (Add/Remove Software or Programs). In the list of installed software look
for items called Windwos Driver Package - Pico uant (P USB). Note, however, that the driver package may
still be needed by other PicoQuant devices. Uninstall it only if you are sure it is no longer required.
Note also that un–installation of the data acquisition software does not automatically uninstall the PQ File Info
shell extension. It can be un-installed as a separate item through the standard Windows Control Panel
mechanisms.
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PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
4. oftware Overview
4.1. tarting the Program
After correct installation the Start Menu Programs contains a shortcut to the HydraHarp software. To start the
HydraHarp software select Pico uant – HydraHarp 400 v3.0.0.1. Note that after switching the HydraHarp on,
you need to allow a warm–up period of at least 20 minutes before using the instrument for serious
measurements. You can use this time for set–up and preliminary measurements.
If the HydraHarp software cannot find the device (or if there are driver problems) it will display a notification
message, but it can still start. Device dependent toolbar buttons and functions of the program will then remain
disabled. This allows you to use the software without the HydraHarp hardware, e.g. to view or print files on
another computer.
If the HydraHarp 400 is correctly installed and there are still device related errors, you can use the Windows
Device Manager for troubleshooting (see the corresponding section above). If problems cannot be resolved,
see section 7 for support. If possible, try the device on another computer.
For regular use of the HydraHarp you may want to create an icon for it on your Windows desktop. If you missed
this during software setup you can do it any tim later via a right mouse click on the start menu entry ( send to >
Desktop – create shortcut). Alternatively use the Windows Explorer to locate the file HydraHarp.exe in the
directory you selected for installation and drag the icon onto the desktop. This will create a link to the
HydraHarp executable file.
You can also start up the HydraHarp software directly from a HydraHarp histogram file by double–clicking on
the file or dragging it onto the HydraHarp icon.
4.2. The Main Window
The HydraHarp software provides a measurement control interface to the HydraHarp hardware and an online
histogram display. The HydraHarp main window accommodates the histogram display area.
Above the display area is the toolbar. Here you can access frequently used commands by simple mouse click.
Above the toolbar is the menu bar for access to additional commands. At the bottom of the histogram display
area is a set of ‘panel meters’ showing count rates, count sums, and histogram peak characteristics. These will
be updated continuously, some only when a measurement is in progress. Note the selector at the right of the
panel meters. This selects the channel the meters are displaying. Instead of a single channel you can also
select Sum, which then displays the summed rates from all channels. The panel meters can be enlarged by
double–clicking, which is useful for optical alignment etc. when the PC is at some distance.
In the top center of the display area a title line is shown. This can be double–clicked to edit the title. When
editing the title, note that only the first line will appear in the display. The remaining lines are meant to be used
as a file comment. All lines will be stored with the file data, but the maximum total is 256 characters.
The main window is resizeable and the actual histogram display will adapt its size accordingly. If you make the
window smaller than the minimum histogram display, two scrollbars will permit access to hidden window areas.
Note that the actual size of the main window depends on the system font selected. It will scale to the size of the
current system font. Screen resolutions under 800x600 are not suitable for serious work with the software.
Note that the position and size of the main window on the screen will be stored in the Windows registry and
retrieved during the next program start. The registry settings are kept separately for each user, provided
he / she is logged on with a unique user name. Consult the section 6.1 for further main window command
descriptions. Toolbar, Menus, panel meters etc. will be explained in the next sections.
At the very bottom of the main window there is a status bar. The leftmost area of the status bar describes
actions of menu items as you navigate through menus. Similarly it shows messages that describe the actions
of toolbar buttons. The second status bar area from the left shows the current measurement status of the
HydraHarp. The rightmost area of the status bar indicates if the <Caps> and <NumLock> keys are latched
down.
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PicoQuant GmbH HydraHarp 400 Software V. .0.0.1
When the HydraHarp software is running with functional hardware it continuously collects
information about the input signals and the current acquisition settings. If these settings together
with the input rates indicate possible errors, the software will activate a warning icon in the status
bar. The warning icon can be clicked to display a list of current warnings together with a brief
explanation of each warning (see also section 8.1).
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