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PicoHarp 300 - Center for Detectors

1. Crv i Curvelndex int32 curve index 0 511 Crv i TimeOfRecording time_t time of recording this curve Crv i Hardwareldent char 16 hardware identifier string Crv i HardwareVersion char 8 hardware version string Crv i HardwareSerial int32 hardware serial number Crv i SyncDivider int32 input channel O Sync input divider Crv i CFDZeroCross0 int32 input channel 0 CFD zero cross level in mV Crv i CFDLevelO int32 input channel 0 CFD discriminator level in mV Crv i CFDZeroCross int32 input channel 1 CFD zero cross level in mV Crv i CFDLevel1 int32 input channel 1 CFD discriminator level in mV Crv i Offset int32 offset in ns Crv i RoutingChannel int32 0 3 Crv i ExtDevices int32 external devices bitwise coded Crv i MeasMode int32 O interactive Crv i SubMode int32 oscilloscope 0 integration 1 TRES 2 Crv i P 1 float used for automated measurements e g TRES nm Crv i P2 float reserved for automated measurements Crv i P3 float reserved for automated measurements Crv i RangeNo int32 O base resol 1 x2 2 x4 3 x8 4 x16 x128 Crv i Resolution float resolution in ns Crv i Channels int32 number of channels bins in this curve Crv i AcquisitionTime int32 Acquisition Time in ms Crv i StopAfter int32 stopped after this many ms Crv i StopReason int32 O timeover 1 manual 2 overflow Crv i InpRateO int32 input O count rate as displayed by rate meter Crv i InpRate1 int32 inpu
2. The following are the T3 records for each i 1 to NumRecords T3Record i uint32 The bit allocation in the record is starting from the MSB channel 4 dtime 12 nsync 16 The channel code 15 all bits ones marks a special record Special records can be overflows or external markers To differentiate this dtime must be checked e Ifitis zero the record marks an overflow e fitis gt 1 the individual bits are external markers See the demo programs for further details end of block These items are stored as they were set displayed last They may not relate to the T3 data stored These items may vary and thereby change the file structure dynamically A program must interpret these before reading in further data See section 8 2 4 for the data types used Note that the T3Records must be read sequentially so that time overflows can be corrected for based on the special records The software setup will install ready to use demos showing how to do this There is a set of examples in C Pascal Delphi Lazarus and Matlab for Pico Harp pt3 file access They can be found in the subfolder Filedemo_ in your chosen installation dir ectory Note that these are only demos The intention is not to routinely convert TTTR data to ASCII This would be highly inefficient It increases the file sizes enormously and adds no useful information TTTR data is not really human readable It requires application specific
3. component 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 instru ment 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 un der favourable conditions lifetimes down to 1 10 of the IRF width FWHM can still be recovered via decon volution 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 con tinuous distribution around any true time value In order to form a histogram at some point the timing results must be quantized This quantization introduces another random error if chosen too coarse The quantiza tion step width i e the resolution must therefore be small compared with the continuous IRF From an in formation theoretical point of view this is governed by sampling theory As a minimum sampling frequency one would therefore assume the Nyquist frequency
4. E 16 206 2 148 WIEN 0 fw E E ER E OR E E F pes P sch e E E E E Es IE DJ IEW E VIEL VIEL la TL Ge SS le a E e len a Ben Elan ae len a ae i i E ba a E E ieee VIEN Close i In the Trace Mapping dialog you can tick the individual boxes Show to display a curve Select the number of the memory block you wish to map the individual display curve to Choose the active memory block you wish to use for the next measurement in the PicoHarp control panel with the Map To edit box and spin control The dialog also provides some statistics on each curve central matrix of figures Resolution is the channel width bin width of the histogram in picoseconds Next is the Max Count the count in the highest point of the curve The column at Time shows the time corresponding to the Max Count channel Leftmost there is the Full Width Half Maximum FWHM of the curve peak usually for IRF traces There are also some buttons for frequently required actions The button view can be clicked to see more detailed curve information such as time of recording acquisition settings and count rates The button All can be clicked to tick all traces as shown The button 0 7 can be clicked to set the default mapping of trace 0 7 to block 0 7 The Clear buttons trash cans can be clicked to delete the contents of individual blocks Note that the Trace Mapping Dialog is non
5. Print the currently displayed histogram curves Activate context help Copy the currently displayed curves to the clipboard ASCII export Launch the axis panel Launch the data cursor dialog Calibrate the time measurement hardware Start measurement based on current PicoHarp control panel settings Stop measurement and histogram accumulation Launch the PicoHarp control panel Launch the trace mapping dialog Launch the TTTR mode dialog Launch the TTTR mode real time correlator dialog Launch the dialog for PHR 800 router setup Launch the dialog for monochromator control and TRES setup 3 5 UBAO AEE DEON gE Launch the software settings dialog Page 18 PicoQuant GmbH PicoHarp 300 software v 2 3 4 4 The Control Panel The PicoHarp control panel is a dialog box for setting the parameters for hardware adjustment and data ac quisition It is implemented as a non modal dialog box i e it does not have to be closed before the main window can continue This way you can make changes to your settings in the control panel and watch their effect on a running measurement in the main window immediately Nevertheless you may close the control panel and restore it at any time by clicking the control panel button on the toolbar or pressing lt A1t gt C Note that the control panel memorizes its last position on the screen even across sessions It will re open where it was last moved PicoHarp Control Panel x Inp
6. TT TR mode only C3 TTL in marker bit 2 input TT TR mode only C4 TTL in marker bit 3 input TT TR mode only C5 GND ground connector DVI I 24 4 Molex 0743232033 available e g from Farnell All ground pins are common ground for the TTL signals and the DC supply lines They are also connected to the chassis Pins 14 provides a 5 V DC supply voltage external electronics can use Under no circumstances must this line be shorted or in total loaded in excess of the specified maximum current Note that the maximum cur rent on the 5 V line must be shared with any loads connected at the 5 V line of the RT connector Pin 8 is a TTL output that indicates that the ctc is active measurement running Note that this is a standard TTL output so it can NOT drive 50 Q loads Pin 16 is pulled high by the PHR 800 to indicate that routing signals are to be accepted through the CTRL connector rather than the RT connector Pins 23 and 24 accept a LVDS signal that is used by the router to mark counts as valid It can also be used to externally enable disable recording of incoming counts This mechanism can be used to blank out periods where e g an external scanner is moving to the next position The count enable signal is effective in all modes It may be used to synchronize the acquisition with external conditions These pins can not be used if a router is connected Page 69 PicoQuant GmbH PicoHarp 300 software v 2 3 Pins C1 through C4 accep
7. The adjustment range is 0 to 800 mV for the CFD discriminator level and 0 to 20 mV for the CFD Zero cross level The initial setting of the discriminator level of 30 mV should be approximately right to suppress any electrical noise If you lower it you should see an increase in count rate probably mostly noise if you raise it you should see a reduced rate Stay closely above the noise level If you still see millions of counts per second at levels over 50 mV reduce the detector illumination Your detector may be at risk at such high rates You can then also try out responsivity to illumination changes The Zero Cross level is not yet meaningful It can be adjusted for optim um timing later To actually collect histograms select a measurement range large enough determined by the chosen re solution and a display range to cover your sync interval i e 1 fsync if possible Initially set Offset O and StopAt 65535 Start a measurement in oscilloscope mode with e g 1 second acquisition time See section 5 2 Setting up and Running Interactive Measurements for how to start a measurement If there are counts coming in you can try to set an upper limit of the count axis so that the histogram is scaled for best viewing If so far you used uncorrelated light you may now want to move to time correlated measurements With a detector signal e g laser induced or a Take electrically derived from the laser sync you can try to obtain a histogram that
8. Use this command to enlarge the active window to fill the available space Note The command is unavailable for already maximized windows Shortcut Mouse Click the maximize button on the title bar or double click the title bar of a not enlarged window Keys lt Alt gt lt Space gt X Restore command Use this command to return from an enlarged window to the previous size Note The command is only available for already maximized or minimized windows Shortcut Mouse Click the maximize button J on the title bar or double click the title bar of the enlarged window Keys lt Alt gt lt Space gt R Close command Use this command to close the active window or dialog box Double clicking a system menu box is the same as choosing the Close command Shortcuts Mouse Click the close button on the title bar Keys lt Alt gt lt F4 gt lt Alt gt lt Space gt C Page 40 PicoQuant GmbH PicoHarp 300 software v 2 3 6 2 Toolbar Toolbar buttons of special interest are explained in some more detail here Context Help When you choose the Context Help button the mouse pointer will change to an arrow and question mark Then click somewhere in the PicoHarp window such as on another toolbar button The help topic will be shown for the item you clicked Ed Axis Settings button Clicking this button opens the Axis Settings Panel This panel provides controls for the axis limits and lin log display switching Double
9. eral rv mai Discr mi ai LED 2 Aero rv m Disecr mi 204 CFO 3 eroacr mi mc Discr mi BE CFO 4 erocr mi E Discr mi 204 T aa The input channel sections provide radio buttons for the input type the level and the active edge For TTL and NIM the input levels and edges are fixed For custom input types they can be adjusted as required Note that it is important to select valid combinations of level and edge Otherwise especially if no detector is con nected the input can be locked in a forbidden state where all other channels will also be locked Please use the standard settings for TTL and NIM unless you really need custom settings If CFDs are used they must be physically connected at the front panel of the PHR 800 The software does not recognize if and how such connections exist Since the software layout suggests a direct match of chan nel number and CFD number it is strongly recommended to connect the physical devices accordingly All other situations would very likely lead to confusion When CFDs are used the corresponding input channels must be set for NIM The CFDs are identical to those of the PicoHarp s own built in CFDs Their operation therefore follows the same guidelines and conventions The adjustment ranges are also the same Note that due to the polarity convention used in the PicoHarp s control panel all CFD settings are shown without sign although the
10. l e 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 no benefit in sampling the histogram at resolutions much higher than 1 10 of the overall IRF of the analog part of the system Nevertheless a good resolution is beneficial in data analysis via iterat ive reconvolution Page 7 PicoQuant GmbH PicoHarp 300 software v 2 3 Photon Counting Detectors Photomultiplier Tube PMT A PMT consists of a light sensitive photocathode 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 col lected at the anode of the PMT and output as a current The current is directly proportional to the intensity of light striking the photocathode 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 var ied to change the sensitivity of the PMT Current PMTs have a wide dynamic range i e they can also meas ure relatively high levels of light They are furthermore very fast so that rapid
11. modal This means the dialog can remain open while a measure ment is in progress so that adjustments can be made under immediate visual control similar to the opera tion of the control panel Note also that a measurement can be running in a block that is not mapped or shown The individual trace mapping dialog items are discussed in section 6 5 Trace Mapping Dialog in the Con trols and Commands Reference Page 21 PicoQuant GmbH PicoHarp 300 software v 2 3 5 Specific Measurement Tasks 5 1 Setting up the Input Channels If you are running TCSPC measurements for the first time we strongly recommend you read our TechNote on TCSPC first The document is provided on your distribution CD It will help you getting a background on the concepts and terms used here In order to acquire any data the two input channels Channel 0 usually sync and Channel 1 usually photon detector of the PicoHarp 300 must be set to match their electrical input signals The PicoHarp input chan nels are designed identically Both are designed as a Constant Fraction Discriminator CFD They can handle an input pulse peak range from 0 to 1 V for regular operation The absolute maximum rating bey ond which damage may occur is 2 V In case of coincidence correlation experiments between two detectors the channels are used for one detect or each In time resolved fluorescence with a pulsed excitation source typically a laser Channel O is used as
12. ous 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 PicoHarp 300 is somewhat different Today it is state of the art that the tasks convention ally performed by TAC and ADC are carried out by a so called Time to Digital Converter TDC These cir cuits allow not only picosecond timing but can also extend the measurable time span to virtually any length by means of digital counters The PicoHarp 300 does not use one such circuit but two They independently work on each input channel 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 con Page 9 PicoQuant GmbH PicoHarp 300 software v 2 3 sists 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 Histogrammer Readout Subtract The full strength of the PicoHarp 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 reconfigu
13. polarity and amplitude The leading edge should be steep there should also be no excessive overshoot or ringing All signals should have rise times of no more than 2 ns Slower signals may not be seen by the PicoHarp and will certainly degrade timing accuracy The pulse width should be 0 5 to 30 ns The upper limit is not critical but the pulse width should be as small as necessary to fit in half the sync period If the signal is satisfactory connect the signal to the Channel 0 input and start the PicoHarp software if it is not yet running A detector signal Channel 1 is not required at this point but it does no harm if it is also connected Open the PicoHarp control panel Leftmost there is a group of controls for Channel O sync Find the edit box and spin control for the CFD discriminator level The discriminator level should be set to a value around half of the amplitude of the sync pulses Note that the control panel shows absolute numbers from 0 to 800 despite the real signals are negative going Next set the Zero Cross level to 10 mV Then look at the Sync Divider It must be set so that the sync rate divided by the shown divider value does not exceed 10 MHz E g for a diode laser with 80 MHz repetition rate it must be set to 8 For a slow source such as a flash lamp it must be set to None Generally it is recommended to use the lowest suitable divider If the sync source is active the Input O rate will now be displayed at the bottom le
14. CFD is also usable with pre shaped signals and simply becomes a level trigger in this case This is the case also for the PicoHarp 300 Note that the input signals must have sufficiently fast rise fall times 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 or der 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 Pro grammable 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 continu
15. The setup program will install the following files in the chosen destination folder e PicoHarp exe the main executable program e PicoHarp chm the PicoHarp compiled help file e Manual pdf the manual in Adobe PDF format e Readme txt a text file with program information e unins000 exe the uninstaller program file e unins000 dat the uninstaller information file Setup will also create a subdirectory DRIVER which contains the device drivers A further subdirectory named FILEDEMO contains demo source code for access to PicoHarp 300 data files in various program ming languages Another folder SAMPLEDATA will be created with samples of PicoHarp data files After the installation the PicoHarp software should be available under the Windows Start Menu under Programs PicoQuant PicoHarp 300 v2 3 or the folder name you chose during setup However be fore running the software please proceed to the hardware installation step Page 14 PicoQuant GmbH PicoHarp 300 software v 2 3 3 3 Device Installation Make sure to avoid electrostatic discharge when handling the PicoHarp 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 To connect the PicoHarp device to the PC first consult your PC manual as to whether and where it provides high speed USB 2 0 connectors The PicoHarp sy
16. These are and must be only about 1 to 5 of the excitation rate and can therefore be handled easily by the TAC ADC or TDC The con sequence of this approach however is that the times measured are not those between laser pulse and cor responding photon event but those between photon event and the next laser pulse This still works by soft ware 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 pos sibly causing trouble when there is no CFD at one of the inputs The PicoHarp 300 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 two 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 lt 1 MHz the sync divider must not be used s
17. WWW http www picoquant com Page 70
18. actu al electrical signals are negative This is different from the router input level setting where both positive and negative signals may occur so that they must be shown accordingly including the sign The router settings are kept in the registry so that at the next start of the software they will still be there Similarly they are kept in the measurement files so that they can be retrieved that way The router settings for each measured curve are also kept in the data files In this case they are only for the routing channel used during the recording of that curve They can be viewed through the Trace Mapping dialog Note that the router settings are ambiguous if a router was used but not enabled routing off The router settings are then marked as na in the curve details view Page 28 PicoQuant GmbH PicoHarp 300 software v 2 3 5 4 Time Tagged Mode Measurements Time Tagged Time Resolved TTTR mode allows the recording of individual count events directly to hard disk without on board forming of histograms It originates from the TimeHarp TCSPC systems and has been further improved for the PicoHarp 300 In TTTR mode in addition to the start stop timing with picosecond resolution the timing of the events with respect to the beginning of the whole measurement is recorded in the event records This mode is particu larly interesting where the dynamics in a fluorescence process are to be investigated The availability of the time
19. analysis software in any case and the demos should be used as a starting point for writing such software Page 62 PicoQuant GmbH PicoHarp 300 software v 2 3 8 2 4 Data Types The PicoHarp software is written in C and the data types used in the files correspond to standard C C data types as follows char byte characters in ASCII int32 32 bit signed integer int64 64 bit signed integer uint16 16 bit unsigned integer uint32 32 bit unsigned integer float 32 bit floating point number time TI a 32 bit time value as defined in most C libraries Note that the formats for date and decimal point may depend on your Windows settings at run time of the PicoHarp software usually national language dependent Note also that on platforms other than x86 machines byte swapping may occur when the PicoHarp data files are read there for further processing We recommend using the native x86 environment consistently The distribution pack includes a set of demo programs source code for various programming languages to show how access to PicoHarp data files can be implemented They will be installed in the subfolder Filedemo under the chosen installation folder Page 63 PicoQuant GmbH PicoHarp 300 software v 2 3 8 3 Hardware Technical Data 8 3 1 All information given here is reliable to our best knowledge However no responsibility is assumed for pos sible inaccuracies or omissions Speci
20. chosen directory filtered by Files of type File name Type or select the filename you want to open This box lists files with the extension you select in the Files of type box Files of type Select the type of file you want to open The default PicoHarp extension is phd Save command Use this command to save the current histogram data to its current name and directory When you save a histogram for the first time PicoHarp displays the Save As dialog box so you can name your file This command is unavailable if a measurement is running If you want to change the name and directory of an ex isting file before you save it choose the Save As command Shortcuts Toolbar Keys SE Le SE lk Save As command Use this command to re name the current histogram data and save The software displays the Save As dialog box so you can name your file To save a file with its existing name and directory use the Save com mand File Save As dialog box The following fields allow you to specify the name and location of the file you are about to save Save in Select i e browse into the directory where you want to store the file Main box In this box you see the content of the chosen directory filtered by Save as type File name Type a new filename to save a histogram with a different name The software auto matically adds the extension you specify in the Save as type box Save as type Selects a filter on the directory
21. details on routing and router connection It also carries marker signals for TTTR mode but these signals can also be fed in via the CTRL connector Therefore if you wish to use rout ing together with marker input the marker signals should be fed in via the CTRL port If necessary a dedic ated Y cable for the simultaneous connection to the RT port can be used available from PicoQuant Note that this cable must not be extended Only required marker signals may be extended to connect the marker source The following table shows the RT connector s pin assignments Pin UO Description 1 TTL in count enable 4 TTL in marker 1 input TTTR mode only 5 TTL in marker 2 input TTTR mode only 9 TTL in marker 0 input TTTR mode only 11 out 5V 0 5V lt 200 mA 12 GND ground 14 GND ground Pin 1 accepts a TTL signal that can be used to externally enable disable recording of incoming counts Counting is enabled when pin 1 is held high This mechanism can be used to blank out periods where e g an external scanner is moving to the next position The count enable signal is effective in all modes It may be used to synchronize the acquisition with external conditions If external disabling is not desired the pin must be held high or left open This signal is used by the router to identify valid counts It can not be used by external applications if a router is connected Pins 4 5 and 9 accept the TTL co
22. 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 PicoHarp 300 has an extremely short dead time of typically 90 ns in fact one of the shortest in any comparable instrument available today Page 6 PicoQuant GmbH PicoHarp 300 software v 2 3 Timing Resolution The most critical component in terms of timing resolution in TCSPC measurements will usually be the detect or 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 T
23. end of block The following is a T2 T3 Mode specific header ExtDevices int32 external devices bitwise coded Reserved int32 reserved Reserved int32 reserved InpRate0 int32 input O count rate as displayed by rate meter InpRate1 int32 input 1 count rate as displayed by rate meter StopAfter int32 stopped after this many ms StopReason int32 O timeover 1 manual 2 overflow NumRecords int32 number of T2 event records ImgHdrSize int32 size of special header to follow before the records start end of block The following is a special header that may not be present at all for each i 0 to lt ImgHdrSize gt Reserved i uint32 reserved end of block The following are the T2 records for each i 1 to NumRecords T2Record i uint32 The bit allocation in the record is starting from the MSB channel 4 time 28 The channel code 15 all bits ones marks a special record Special records can be overflows or external markers To differentiate this the lower 4 bits of time must be checked e If they are all zero the record marks an overflow e If they are gt 1 they are external markers See the demo programs for further details end of block These items are stored as they were set displayed last They may not relate to the data stored These items may vary and thereby change the file structure dynamically A program must interpret these
24. from this comparison changes its polarity exactly when a constant fraction of the detector pulse height is reached The zero cross ing point of this signal is therefore suitable to derive a timing signal independent from the amplitude of the in put 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 PicoHarp models 2 0 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 fur thermore permit setting of a discriminator threshold that determines the lower limit the detector pulse amp litude 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 on both in put channels if e g coincidence correlation experiments between two detectors are carried out A suitably designed
25. from your optical table Input Troubleshooting Whenever there is a problem first check your cabling detector power supply and sync source To be abso lutely sure check the signals with a fast oscilloscope 50 Q input Never try to deliver the signals to multiple 50 Q loads in parallel using simple T pads If you have a router for multiple multiple detectors try to test your setup without the router first but note the different signal levels If you cannot get a stable sync rate reading of expected figures there may be several reasons there is no proper sync signal voltage polarity pulse width frequency e the Channel 0 CFD level setting is inappropriate the sync divider setting is inappropriate Note that at small rates the meter display may fluctuate between two discrete values Page 23 PicoQuant GmbH PicoHarp 300 software v 2 3 Reasons for zero Channel 1 counts are no or inappropriate signal voltage polarity pulse width frequency e inappropriate CFD levels e preamplifier or detector failure router connected but not used bypassed Note that all meters are updated at the display refresh rate it can be set between 0 1 and 1 s for integration mode and it equals the acquisition time in oscilloscope mode In oscilloscope mode if the time is chosen very long this may lead to apparently nothing happening for accordingly long times The refresh rate for In tegration mode can be selected in the Soft
26. has started Note that this is a standard TTL output so it can NOT drive 50 Q loads All other pins are used for communication with dedicated accessories or future extensions and must not be connected otherwise Page 67 PicoQuant GmbH PicoHarp 300 software v 2 3 8 3 2 2 PicoHarp 300 Hardware Version 2 0 For the benefit of technical progress the PicoHarp hardware is undergoing further development Revi sion 2 0 has two new connectors One is a 9 pin connector for CAN bus devices The other is a new differen tial routing and control port connector that replaces the old 26 pin control port connector It carries the rout ing information as differential signals thereby improving signal integrity and reducing crosstalk The CAN connector is designed for PicoQuant accessories such as monochromator stepper motor control lers It must not be used for any other purposes If custom CAN nodes are to be controlled suitable software support is also required so that we recommend contacting PicoQuant for a tailored solution The figure below shows the pin layout of the PicoHarp 300 router control ports as of PicoHarp hardware rev 2 0 There is a 15 pin male sub D connector labeled HI This is the connector primarily for the PHR 402 403 routers The larger DVI I connector labeled CTRL is for generic control signals as well as routing with the PHR 800 Note that several signals are present on both connectors wired through but NOT on the same
27. in the TTTR mode dia log The icon can be clicked to display a list of current warnings together with a brief explanation of each warning Note that the software can detect only a subset of all possible error conditions It is therefore not safe to as sume all is right just by seeing no warning On the opposite if any of the warnings turns out to be an unne cessary nuisance e g because your specific measurement conditions knowingly force it you can disable that warning via the software settings dialog available through the toolbar see section 6 2 The warnings are to some extent dependent on the current measurement mode Not all warnings will occur in all measurement modes Also count rate limits for a specific warning may be different in different modes The following table lists the possible warnings in the three measurement modes and gives some explanation as to their possible cause and consequences Warning Histo Mode T2 Mode T3 Mode WARNING_INPO_RATE_ZERO No counts are detected at input channel 0 In histogramming d d and T3 mode this is the sync channel and the measurement will not work without that signal WARNING_INPO_RATE_TOO LOW The pulse rate at input channel O is below 1 kHz and the sync divider is gt 1 In histogramming and T3 mode this is the d d sync channel and the measurement will not work without a signal lt 1 kHz if the sync divider is gt 1 If your sync rate is really so low then you do not need a
28. is 3 in T3 Mode Format version 2 0 pt3 files are structured as shown in the table below Note that the format version is not necessarily the same as that of the creating software version Data Item Type Description Ident char 16 the string PicoHarp 300 FormatVersion char 6 currently 2 0 since Sept 2006 CreatorName char 18 for this software PicoHarp Software CreatorVersion char 12 currently 2 3 0 0 April 2009 File Time char 18 the file creation date and time CR LF char 2 Ox0D 0x0A carriage return line feed Comment char 256 any ASCII string NumberOfCurves int32 not meaningful in T3 Mode BitsPerRecord int32 32 RoutingChannels int32 1 or 4 NumberOfBoards int32 reserved now 1 ActiveCurve int32 curve no used for last measurement MeasurementMode int32 O interactive 1 reserved 2 T2 3 T3 SubMode int32 O oscilloscope 1 integration 2 TRES RangeNo int32 O base resol 1 x2 2 x4 3 x8 4 x16 x128 Offset int32 offset in ns AcquisitionTime int32 acquisition time in ms StopAt int32 stop at this many counts StopOnOvfl int32 stop if overflow occurred 1 otherwise 0 Restart int32 restart 1 otherwise 0 DisplayLinLog int32 lin 0 log 1 DisplayTimeAxisFrom int32 lower time axis bound of display in ns Display TimeAxis T 0 int32 upper time axis bound of display in ns DisplayCountAxisFrom int
29. location of your choice and run the setup from there Note that if the distribution was by email the installer file may have been renamed to setup ex__ in order to let it pass Strict email filters In that case please rename it back to setup exe In order to use the PicoHarp software with the PicoHarp 300 hardware a suitable device driver must be in stalled The PicoHarp 300 is a USB 2 0 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 prompt the user for the appropriate driver disk Note that the software setup for the PicoHarp 300 preinstalls the driver so that upon connecting the device it is readily available for the Win dows plug amp play device management and you should not be prompted for the driver location This requires that the software setup is performed before connecting the device Dependent on your version of Windows you may still be prompted to confirm the final driver installation Some windows versions verbosely warn about drivers not being validated by Microsoft You can ignore these warnings When upgrading from an earlier version of the PicoHarp software on a machine where the device driver is already installed you may also want to perform a driver update via the Windows device manager For that purpose the device must be connected but no software should be using it On some W
30. of current warnings together with a brief explanation of each warning see also section 8 1 Note that the software can detect only a small subset of possible error condi tions It is therefore not safe to assume all is right just by seeing no warning On the opposite if any of the warnings turns out to be an unnecessary nuisance e g because your specific measurement conditions will expectedly cause it you can disable that warning via the software settings dialog available through the tool bar see section 6 2 Page 24 PicoQuant GmbH PicoHarp 300 software v 2 3 5 2 Setting up and Running Interactive Measurements If you are running TCSPC measurements for the first time we strongly recommend you read our TechNote on TCSPC first See your distribution CD This will help you getting started much quicker This manual can not replace a textbook To set up measurement parameters use the PicoHarp control panel The control panel can be opened by clicking the control panel button on the toolbar or by pressing lt ALT gt C In the control panel section Acquisition you can set the resolution time per bin the offset the measurement time and the block of memory to use for this measurement To begin use a measurement time of 1 second and an offset of 0 There are always 65536 time channels bins per PicoHarp histogram Histograms can be measured in 512 memory blocks Out of these up to 8 curves can be displayed and one
31. or the Apply button The spin controls can be used to increment or decrement the edit box values In this case the changes take effect immediately without need for lt Enter gt or Apply Check boxes have their denoted effect when the tick is shown They can be toggled with a mouse click Groups of radio buttons are like check boxes but mutually exclusive They work like on an old radio one that was pressed pops out when another one is pressed Note that the settings of the axis panel as well as the positions of the panel on the screen will be stored in the Windows registry and retrieved during the next program start The registry settings are kept separately per user The individual axis panel items are discussed in the section 6 4 Axis Panel in the Controls and Commands Reference To begin use logarithmic display to make sure even weak signals can be seen Page 20 PicoQuant GmbH PicoHarp 300 software v 2 3 4 6 The Trace Mapping Dialog The PicoHarp software can measure histograms in up to 512 memory blocks Out of these up to 8 curves can be displayed The Trace Mapping dialog is used to select the curves to display It also allows to view curve details and cleanup You can use the Trace Map button on the Toolbar or click the curve colour indic ator in the control panel to launch the dialog PicoHarp Trace Mapping l x Trace Show MapTo Resolution ps Max Count at Time na FWHM ns Details 4 10000 15 368 0 260 VIEW
32. records so that excess marker traffic can suppress photon records In fast imaging applications it is therefore recommended not to use a pixel clock but a line clock only Because each photon has a time tag it is usually not necessary to use an additional pixel clock There is another TTL input that can be used to externally enable disable the recording of incoming counts see section 8 3 2 Counting is enabled when this input is held high This mechanism can be used to blank out periods where e g an external scanner is moving to the next position The count enable signal is also active in interactive mode It may be used to synchronize the start of acquisition with external events If ex ternal disabling is not desired the pin must be held high or left open Note that if a router is connected this pin is not available for user signals 1 not available for PicoHarp 300E Page 32 PicoQuant GmbH PicoHarp 300 software v 2 3 A TTL output ctc active is provided to signal the start of a measurement This can be used to notify or trig ger other hardware If you wish to use markers the marker signals must be fed in via the CIR or RT port see section 8 3 2 If routing is used together with markers the free port can be used for feeding in markers lf necessary a dedicated Y cable for the simultaneous connection to the RT port can be used available from PicoQuant Note that this cable must not be extended Only required marker signals m
33. starting a TRES measurement will clear and subsequently overwrite all curves in memory that may have been recorded previously If the curves in memory were loaded from a file the actual file on disk will not be overwritten until you save to the file Save menu or corresponding toolbar button Use Save As and select a new file name if you want to preserve the old file The TRES measurement is selected in the PicoHarp control panel You need to use the selection box for the measurement mode to select TRES mode instead of OSC or INT Acquisition Resol ps Offset n d l Routed T Restart T Stop Of Black A TRES measurement is started or canceled as usual via the toolbar buttons for start and stop The data acquisition will be performed like a standard Integration Mode measurement while the block number is incremented for each new wavelength At the beginning of each TRES run the active curve block will be set to 0 or to 1 if no IRF is collected All curves that were previously collected and not saved to a file will be overwritten with new data During a TRES run the currently collected data will always be shown as Trace O dark blue even though the block Trace number is actually incremented at each wavelength step This is to overcome the limitation of only 8 display curves being available You can watch the block number in the control panel to see the curve cur rently being collected Manual entry will be disabled
34. successive events can be reli ably 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 photocathode 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 differenti ate between individual pulses and the photon counting detector will become non linear This usually occurs at 1 to 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 histo grams 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 effi ciency in the blue green region depending upon photo cathode materials Typical maximum quantum effi ciencies are about 25 For spectroscopy experiments in the ul
35. tags permits photon burst identification which is of great value e g for Single Molecule Spectroscopy SMS in a liquid flow Other typical applications are Fluorescence Correlation Spectroscopy FCS and Burst Integrated Fluorescence Lifetime BIFL measurements Together with an appropriate scan controller TTTR mode is also suitable for ultra fast Fluorescence Lifetime Imaging FLIM Applications beyond fluores cence spectroscopy are e g in quantum information processing notably quantum key distribution and re lated basic rearch The PicoHarp 300 actually supports two different Time Tagging modes T2 and T3 mode which will be ex plained further below When referring to both modes together we use the general term TTTR here TT TR mode measurements always implicitly provide routing information if a router is used Routing informa tion is represented by four bits in each TTTR record that allow up to four routing channels to be distin guished However these bits are only meaningful if a router was actually used for the measurement Routing will probably not be useful in T2 mode where the main objective is typically coincidence correlation With all current routers only two routing bits are used 4 channels Throughput constraints and crosstalk considera tions apply the same way as in interactive routed measurements Note TTTR mode is not part of the standard PicoHarp product It must be purchased as a separate firmware option that gets b
36. the printer and its connection Shortcut Keys lt A1t gt F R 1 2 3 4 command most recently used files Use the numbers and filenames listed at the bottom of the File menu to open the last four files you closed Choose the number that corresponds with the file you want to open Shortcuts Keys lt A1t gt F 1 lt AILt gt F 2 lt ALt gt t F 3 lt AIt gt F 4 Exit command Use this command to end your PicoHarp session Save your data before exiting Shortcuts Mouse Click the close button I on the title bar Keys lt Alt gt lt F4 gt SEL dek X Page 48 PicoQuant GmbH PicoHarp 300 software v 2 3 6 6 2 Edit Menu This menu offers only one command Copy command Copies the currently displayed curves in ASCII format to the clipboard This can be used to export histogram data to spreadsheet or data analysis software e g the FluoFit Global Fluorescence Decay Fit Software Note that only the currently selected curves within the current display limits are copied If the curves in the display are of different resolution then all 65536 channels will be copied Copying data to the clipboard re places the contents previously stored there The exported data is plain lt Tab gt separated ASCII and can be read by most of the common spreadsheet programs or data analysis software This command is unavailable while a measurement is running Shortcuts Keys lt Alt gt E C SCEE de 6 6 3 View Menu The View m
37. 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 PicoHarp All signals should have rise times of no more than 2 ns Slower signals may not be seen by the PicoHarp and will cer tainly degrade timing accuracy When using the PicoHarp 300 in combination with the PDL 800 B the sync pulses from the laser driver should be attenuated by 10 to 15 dB to fall in the optimum range for smallest crosstalk The PicoHarp hardware is offered in two versions The standard product PicoHarp 300 provides more fea tures The PicoHarp 300E is a leaner variant targeting cost sensitive applications where some functionality is not required The features that distinguish the PicoHarp 300 from the 300E model are support for routing devices for simultaneous measurements with several detectors and interfacing with external hardware via TTL marker signals The reduced functionality is physically evident through the presence of two multi pin connectors at the PicoHarp 300 front panel that are not present at the 300E model If these connectors are present do not connect anything other than dedicated hardware to these connectors They are provided for hardware expansion notably routing and experiment control and must not be used otherw
38. 1 integration 2 TRES RangeNo int32 O base resol 1 x2 2 x4 3 x8 4 x16 x128 Offset int32 offset in ns AcquisitionTime int32 acquisition time in ms StopAt int32 stop at this many counts StopOnOvfl int32 stop if overflow occurred 1 otherwise 0 Restart int32 restart 1 otherwise 0 DisplayLinLog int32 lin 0 log 1 Display TimeAxisFrom int32 lower time axis bound of display in ns DisplayTimeAxisTo int32 upper time axis bound of display in ns DisplayCountAxisFrom int32 lower count axis bound of display DisplayCountAxisTo int32 upper count axis bound of display The following block will appear in the file for each i 1 to 8 DisplayCurve i MapTo int32 number of memory block to map this curve to DisplayCurve i Show int32 O don t show 1 show end of block The following block will appear in the file for each i 1 to 3 Param i Start float used for automated measurements e g TRES Param i Step float reserved for automated measurements Garam End float reserved for automated measurements end of block RepeatMode int32 reserved for automated measurements RepeatsPerCurve int32 reserved for automated measurements RepeatTime int32 reserved for automated measurements Page 56 PicoQuant GmbH PicoHarp 300 software v 2 3 RepeatWaitTime int32 reserved for automated measurements ScriptName c
39. 32 lower count axis bound of display DisplayCountAxisTo int32 upper count axis bound of display The following block will appear in the file for each i 1 to 8 DisplayCurve i MapTo int32 number of memory block to map this curve to DisplayCurve i Show int32 O don t show 1 show end of block The following block will appear in the file for each i 1 to 3 Param i Start float used for automated measurements e g TRES Param i Step float reserved for automated measurements Param i End float reserved for automated measurements end of block RepeatMode int32 reserved for automated measurements RepeatsPerCurve int32 reserved for automated measurements RepeatTime int32 reserved for automated measurements RepeatWaitTime int32 reserved for automated measurements ScriptName char 20 reserved for automated measurements The following block will appear in the file for each i 1 to lt NumberOfBoards gt currently this must be 1 Brd i Hardwareldent char 16 currently PicoHarp 300 Brd i HardwareVersion char 8 currently 1 0 or 2 0 Brd i HardwareSerial int32 hardware serial number Brd i SyncDivider int32 input channel 0 Sync input divider Page 61 PicoQuant GmbH PicoHarp 300 software v 2 3 Brd i CFDZeroCross0 int32 input channel 0 CFD zero cross level in mV Brd i CFDLevel0 int32 input chann
40. 5 F Warm up period 220 min Bus interface USB rev 2 0 Recommended PC specs CPU clock 21 GHz RAM 512 MB Operating system Windows 2000 XP Vista Power Consumtion Line 100 V 240 VAC lt 25W Dimensions Table top rack 310 x 235 x 140 mm Retraction of old Devices Waste electrical products must not be disposed Ds of with household waste This equipment should be taken to your local recycling centre for safe treatment WEEE Reg Nr DE 96457402 1 Trademarks of Microsoft Corp Page 65 PicoQuant GmbH PicoHarp 300 software v 2 3 8 3 2 Router Control Port Connectors Note The low cost model PicoHarp 300E does not have these connectors 8 3 2 1 PicoHarp 300 Hardware Version 1 0 The figure below shows the pin layout of the PicoHarp 300 router control ports as of PicoHarp 300 hardware rev 1 0 There is a 15 pin male sub D connector labeled HI This is the connector primarily for the PHR 402 403 routers The larger female 26 pin connector labeled CTRL is for generic control signals Note that several signals are present on both connectors wired through but NOT on the same pin numbers CXXXIII 8 0e00e00 0 ALALIIT LEE E EE WM 1 00 01 RT PicoHarp 300 rev 1 0 router and control connectors viewed from the outside of the chassis RT Connector The 15 pin male sub D connector labeled RT is the connector for the PHR 402 403 routers See section 5 3 on Routed Measurements for
41. 6 channels in one histogram With the different resolutions the approximate time ranges covered are between 65536 4 ps 262 ns up to 65536 512 ps 33 5 us The choice of range must compromise between resolution and time span covered The smallest range offers the best resolution and the shortest span vice versa for the largest range For sync rates gt 5 MHz the highest resolution is usually most appropriate since the smallest range still covers the full sync period For lower sync rates the histogram range is too small to cover the full sync period The decay curve region of interest may therefore lie outside the acquisition window of 65536 channels Apart from switching to a lower resolution it is possible to shift the acquisition window relative to the sync frame by means of the offset Note that this offset cannot replace cable delays for appropriate placement of the signal peak within in the sync span Offset edit box and spin control For sync rates gt 5 MHz the highest resolution is usually most appropriate since the smallest range still cov ers the full sync period The offset should in these cases always be set to 0 For lower sync rates the histogram range may be too small to cover the full sync period The decay curve re gion of interest may therefore lie outside the acquisition window of 65536 channels Apart from switching to a lower resolution it is possible to shift the acquisition window relative to the sync frame by means of set
42. Chan 0 Sune Inp Chan 17 m Acquisition erolr m Divider eral mi nah ul sz Dieser mi Diser mw ez ez Resol ps Offset ne mg a S PR Routed Tro Block Time Stop at Ste WAT WM JI am Si 0000 i Stop OF Apply Input Channel 0 Sync Zero Cross edit box and spin control Discriminator Level edit box and spin control sync Divider edit box and spin control Input Channel 1 Zero Cross edit box and spin control Discriminator Level edit box and spin control Acquisition Resolution edit box and spin control Offset edit box and spin control Time edit box and spin control Trc Block trace colour indicator edit box and spin control Stop At edit box and spin control Mode selection box OSC INT TRES Routed check box Restart check box Stop on Overflow check box The edit boxes are for keyboard entry The values must be confirmed with either the lt Enter gt key or the Apply button The spin controls can be used to increment or decrement the edit box values In this case the changes take effect immediately without need for lt Enter gt or Apply Check boxes have their denoted effect when the tick is shown They can be toggled with a mouse click Groups of radio buttons are like check boxes but mutually exclusive Note that the settings of the control panel as well as the positions of control panel and main window on the screen will be stored in the Windows registry and retrieved during the next progr
43. If this box is ticked checked the measurement will stop on overflow of any histogram bin The overflow limit is set by the StopAt parameter Toggle the current setting with a mouse click Oscilloscope Mode selection box Mode The selection Oscilloscope from the selection box Mode selects the current acquisition mode Oscilloscope for the PicoHarp In oscilloscope mode the acquisition and display once started repeats at intervals given by the current acquisition time setting The histogram accumulation always starts from scratch This is useful for monitoring fast changes during optical alignment etc Integration Mode selection box Mode As opposed to oscilloscope mode the histogram acquisition in integration mode is not reset to zero with each display refresh The histogram continues to grow while the display is updated every 0 1 to 1 seconds determined by the refresh rate value selected in the Software Settings Dialog Acquisition can be manually started and stopped additionally the option stop on overflow will stop the acquisition when the maximum count of any channel overflows The option restart will cause the acquisition to start again after the acquisi tion time has elapsed This is similar to oscilloscope mode but allows watching the histogram grow if the ac quisition time is fairly long Page 44 PicoQuant GmbH PicoHarp 300 software v 2 3 TRES Mode selection box Mode Selects the measurement mode TRES Time
44. N Gpecha 36 6 Controls and Commands ReferenCe ssssssnnnensnnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn ennnen enema 40 SC D EVNA OWN EE 40 Oea EOOD EE 41 COO EE 42 oA PAIS E EE 45 60 9 Trace eeler EE 46 GGs NIO gt ee ee ee E eee E ee eee 46 Oc TS EE 46 OZ TE IS gege 49 E WS NEN EE 49 E FISD INS E 50 6 7 Title and Comment Edor occ ccccccccceececeeeeee cece eeseeeseeeeseeeceaeeseeeesaeessueeeeeesauseseeesgeseegeeeeeseeaes g1 00 Prin Preview Dialogi EE 51 Problems Tips amp reese 52 i ADDEN E 54 Sl A a E E E A E TE E E E E EE E E 54 9 2 Data File FOMMalS nesenie a ee AE OE EA EEEE 56 PicoQuant GmbH PicoHarp 300 software v 2 3 8 2 1 Interactive Mode tietormmat A 56 8 2 2 T2 Mode File tommat 59 EE T3 Mod Hle Tt EE 61 BZA Da KE 63 8 3 Hardware Technical DAA saszivedcocdnies snexeewndedcenseasendonnsneteswadasdduedaneddavayeceawnstasdeeseasasdeceeesdeceygandeensnndeutds 64 8 3 1 COMMONM SPECICATION ER 64 8 3 2 Router Control Port Connechors 66 8 3 2 1 PicoHarp 300 Hardware Version U N 66 8 3 2 2 PicoHarp 300 Hardware Version ZU 68 Page 3 PicoQuant GmbH PicoHarp 300 software v 2 3 1 Introduction While fluorescence spectroscopic investigations are fairly common today extracting additional temporal in formation from molecules via laser induced fluorescence is a relatively new and much more powerful tech nique The temporal analysis can reveal information about the
45. PicoHarp 300 Picosecond Histogram Diff Accumulating Real time Processor EH Time Correlated single Photon Counting System with USB Interface User s Manual and Technical Data Software Version 2 3 PicoQuant GmbH PicoHarp 300 software v 2 3 Table of Contents WT d Te HO TE 4 2 Primer on Time Correlated Single Photon Counting ccccssseeessseeeeeeeseneeeeeneeeeeeeseeneseeseeseneneesenees 5 3 Hardware and Software Installation ccc ceeee eee RRE ERR ENER EE ENER EE RRE KEEN REENEN REENEN ENEE 13 3 1 General Installation Notes Read hett 13 3 2 Software Installaton cc ccccecccseceseeeceeeeeeeeeeeeseeeeceeeeseeeseeeesseeeseeeseeeesaeeseeeeseeesesseeseeaeeseeaeegeeseees 14 3 3 Device INSTANT OM E 15 3 4 Installation Troublesbootmg nennen 16 3 5 Uninstalling the GEDEReesesseegeh deeg GENEE 16 A SONWANE e ENT 17 AT SaN De Fro AN orrea E R EE E E N E as 17 A2 Poe Mam WR lee Te 17 Ao TAE TOODI E 18 AG TNE Control Pane EE 19 do EE ea A A E EE E 20 46 The Trac Mapping D Ielegte tssregegegegeeee dee eu EEN 21 5 Specific Measurement Teens Seege Eege 22 5 1 Setting Up the input Channels siessen aaiae aE E a E eda aAA 22 5 2 Setting up and Running Interactive Meaeurements 25 5 3 Routed Meaeuremente A 26 5 3 1 General Setup and ODperaton 26 592 Usno Ne PHR 909 e EE 28 5 4 Time Tagged Mode Measurements 29 5 5 TTTR Measurements with Real Time Correlatnon 34 5 6 Time Resolved EMISSIO
46. Resolved Emission Spectra This requires a monochromator and corresponding driver hardware software to be installed See section 5 6 on Time Resolved Emission Spectra Stop At edit box and spin control Here the stop count level for histogramming can be set The setting is only meaningful if the check box Stop Ofl is active Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment or decrement the current value In this case changes take effect immediately without pressing lt Enter gt The minimum is 0 If the entered value exceeds the allowed maximum of 65536 an error message appears When using the spin control exceeding the maximum is simply not allowed In stead of incrementing the value further the system beeps a warning 6 4 Axis Panel Time Axis Minimum edit box and spin control Here the start of the displayed time axis can be set Units are nanoseconds ns Type the value as an in teger in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment or decre ment the current value In this case changes take effect immediately without pressing lt Enter gt The minim um is O If the entered value exceeds the current time axis maximum an error message appears When us ing the spin control exceeding the current time axis maximum is simply not allowed Instead of incrementing the minimum value further the system beeps a
47. T TR mode concept as it was first conceived for the TimeHarp system The event record is then composed of two timing figures 1 The start stop timing difference between the photon event and the last sync event and 2 the arrival time of the event pair on the overall experiment time scale the time tag The latter was originally obtained from an independent asynchronous clock This made it difficult to combine the start stop timing with the time tag or to know the sync period the event belonged to For the PicoHarp s T3 mode a smarter approach was chosen The time tag is now obtained by simply counting sync pulses From the T3 mode event records it is therefore possible to precisely determine which sync period a photon event belongs to Since the sync period is also known precisely this furthermore allows to reconstruct the ar rival time of the photon with respect to the overall experiment time Each T3 mode event record consists of 32 bits There are 4 bits for the channel number 12 bits for the start stop time and 16 bits for the sync counter If the counter overflows a special overflow marker record is inser ted in the data stream so that upon processing of the data stream a theoretically infinite time span can be recovered Only for practical reasons of the current software design the number of records is limited to 2147483648 which corresponds to 8 GBytes of data The 12 bits for the start stop time difference cover a time span of 4096 R wh
48. This allows powerful new application such as FCS with lag times from picoseconds to hours Autocorrelations can also be calculated at the full resolution but of course only starting from lag times larger than the dead time Page 29 PicoQuant GmbH PicoHarp 300 software v 2 3 The 32 bit event records are queued in a FIFO First In First Out buffer capable of holding up to 256 k event records The FIFO input is fast enough to accept records at the full speed of the time digitizers up to 10 Mcps each This means even during a fast burst no events will be dropped except those lost in the dead time anyhow The FIFO output is continuously read by the host PC thereby making room for fresh incoming events Even if the average read rate of the host PC is limited bursts with much higher rate can be recorded for some time Only if the average count rate over a long period of time exceeds the readout speed of the PC a FIFO overrun could occur In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained However on a modern and well configured PC a sustained average count rate of 5 Mcps is possible This total transfer rate must be shared by the two input channels For all practic ally relevant fluorescence detection applications the effective rate per channel is more than sufficient For maximum throughput T2 mode data streams are normally written directly to disk There is no immediate data visualization duri
49. act easy to use system with Universal Serial Bus USB interface Its new solid state design allows innovative measurement approaches improves reliability and simplifies calibration The circuit allows high measurement rates up to 10 million counts per second Mcps and provides a time resolu tion of 4 ps The modern USB 2 0 interface provides high throughput as well as plug and play installation The input triggers are programmable for a wide range of input signals Both have a programmable Constant Fraction Discriminator CFD These specifications qualify the PicoHarp 300 for use with most common single photon detectors such as Single Photon Avalanche Photodiodes 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 PicoHarp 300 is perfectly matched to these detectors and the overall Instrument Response Function IRF can be as short as 30 ps FWHM Similarly inexpensive and easy to use diode lasers such as the PDL 800 B with interchangeable laser heads as well as the most ex pensive mode locked lasers can be used as an excitation source This permits lifetime measurements down to under ten picoseconds with deconvolution e g using the FluoFit Global Fluorescence Decay Fit Software The PicoHarp 300 can operate in various modes to adapt to different measurement needs The standard his togram mode performs real time histog
50. acters 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 your PicoHarp main window depends on the system font selected It will scale itself to the size of the current display font On systems with display resolutions of less than 800x600 the full window may fit on the screen only if the small display font is selected Screen resolutions under 800x600 are not suitable for serious work with the program 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 Consult the section 6 1 Main Window for further 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 ac tions 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 Pi coHarp Next to it you may find a warnings icon that you can click for details see also section 8 1 When the PicoHarp software is running with functional hardware it continuously collects in
51. active curve can be used for a measurement You can designate the active memory block you wish to use for the next measurement by selecting the block number in the control panel Never forget to select a new block when collecting new data and old data is intended to be preserved Use the trace mapping dialog to select up to 8 curves for display Make sure the memory block you measure into is mapped to a display trace that is switched on so that you can see the trace You can reach the trace mapping dialog from the toolbar or by clicking on the trace colour indicator next to the block selector The colour indicator shows the trace colour the chosen block is currently mapped to If it is a solid square the curve is mapped and shown If it is mapped but not shown the indicator shows a small striped square If the curve is not even mapped for display the indicator remains white To run a measurement you will need to set up the appropriate CFD levels as outlined in section 5 1 Setting up the Input Channels There are two basic histogramming modes for interactive measurements Oscilloscope and Integration mode Oscilloscope mode repeatedly collects histograms with a fixed measurement time and displays them on the screen This lets you see fast changes in the histogram e g for optical setup and adjustments Usually this only makes sense with relatively strong signals and short acquisition times Integration mode is usually operated with longer acqui
52. am start The registry set tings are kept separately per user When a PicoHarp data file is loaded the control panel settings will change according to the settings in that file The individual control panel items are discussed in the section Control Panel in the Controls and Commands Reference Page 19 PicoQuant GmbH PicoHarp 300 software v 2 3 4 5 The Axis Panel The axis settings panel is a dialog box for setting the axis range for the histogram display in the main win dow It is implemented as a non modal dialog box i e it does not have to be closed before the main win dow can continue This way you can make changes in the axis panel and watch their effect in the main win dow immediately Nevertheless you may close the axis panel and restore it at any time by clicking the axis panel button on the toolbar or pressing lt ALT gt A The panel will also open if you double click the axes in the main window x Time Axis Count Axis dm ns ax ns 100 GC The axis panel consists of two groups containing edit boxes and other controls for related parameters These groups and their respective controls are Time Axis Minimum edit box and spin control Maximum edit box and spin control Count Axis Minimum edit box and spin control Maximum edit box and spin control Linear radio button Logarithmic radio button The edit boxes are for keyboard entry The values must be confirmed with either the lt Enter gt key
53. and permits an instrument response of lt 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 102 from PicoQuant This gives pulses of 100 to 500 mV Cabling is double shielded 50 Q coax cable If the detector is a SPAD module with TTL output e g SPCM AQR from Perkin Elmer then an inverter attenuator SIA 400 must be inserted The PDL 800 B laser driver directly 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 Q coax cable When using the PicoHarp 300 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 out put of the laser driver This reduces crosstalk into the relatively weak detector signals r sample Cuvette Cutoff Filter Neutral Density Filter Laser Head USB Ch 1 photon POL 800 6 Diode Laser Driver FicoHarp 300 Do syncout 40 dB Ch O nc 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 followi
54. 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 conventional 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 mo lecule 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 im portant 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 3 to 5 Mcps With the PicoHarp 300 average count rates of 5 Mcps and peak rates up to 10 Mcps can be collected It is worth not ing that the count arrival times are typically random so that there can be bursts of high count rate and peri ods 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 in terpret their maximum count rates
55. ay be extended to connect the marker source The PHR 800 has its own marker inputs so that simultaneous connection to the CIR port of a PicoHarp model 2 0 should never be necessary Using TTTR Mode Data Files For diagnostic purposes you may re load a T3 mode file into the PicoHarp software The limitation is that you will only be able to form a histogram over the start stop times in your T3 mode data The time tag information will not be used here The PicoHarp software will recognize that you are loading a T3 mode file and how many records there are contained in it It will then prompt you for a range to use for histogramming Make sure to keep this range limited to a reasonable size since the histogram channels are limited so overflow may occur The histogram will go to block O of the histogram memory A TTTR mode file also contains all control panel settings that were active at the time of the measurement run However it does not contain any count rate information or pulse FWHM This is why the respective meters will show zero after loading such a file 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 phd 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 interac
56. before reading in further data See section 8 2 4 for the data types used Note that the T2Records must be read sequentially so that time overflows can be corrected for based on the overflow records The software setup will install ready to use demos showing how to do this There is a set of examples in C Pascal Delphi Lazarus and Matlab for Pico Harp pt2 file access They can be found in the subfolder Filedemo_ in your chosen installation direct ory Note that these are only demos The intention is not to routinely convert TTTR data to ASCII This would be highly inefficient It increases the file sizes enormously and adds no useful information TTTR data is not really human readable It requires application specific analysis software in any case and the demos should be used as a starting point for writing such software Page 60 PicoQuant GmbH PicoHarp 300 software v 2 3 8 2 3 T3 Mode File Format PicoHarp data files from T3 Mode pt3 are binary files which contain both the setup parameters and the actual event data The setup data is in the file header which is in the same format as in standard interactive mode and T2 Mode files This is to allow common reading routines for the headers Note that this leads to some header values that are not meaningful in T3 Mode In order to safely recognize a T3 Mode file apart from the file extension a program reading in these files must read and interpret the value MeasurementMode which
57. box and spin control Here the start of the displayed count axis can be set Units are counts Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the current value In this case changes take effect immediately without pressing lt Enter gt In linear scale mode the de fault increment decrement is 100 In log scale mode it is one decade If the entered value exceeds the cur rent count axis maximum an error message appears When using the spin control exceeding the current count axis maximum is automatically prevented Instead of incrementing the minimum value further the sys tem beeps a warning Count Axis Maximum edit box and spin control Here the upper end of the displayed count axis can be set Units are counts Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment or decrement the current value In this case changes take effect immediately without pressing lt Enter gt In linear scale mode the default increment decrement is 100 In log scale mode it is one decade If the entered value becomes smaller than the current time axis minimum an error message appears When using the spin control violat ing the current count axis minimum is prevented automatically Instead of decrementing the minimum value further the system beeps a warning Page 45 PicoQuant GmbH PicoHarp 300 so
58. chosen in the Save in box Only files that pass the fil ter are shown in the dialogs main box Additionally the software adds the extension associated with the chosen filter to a given file name if it was given without exten sion Page 47 PicoQuant GmbH PicoHarp 300 software v 2 3 Print command Use this command to print the currently displayed histogram curves This command presents a Print dialog box where you may specify the number of copies the destination printer the paper orientation and other printer setup options Shortcuts Toolbar Keys lt Ctr1l gt P aii E Page Setup command Use this command to change the print layout This command presents the page setup dialog box where you may select or deselect various items to appear in the print You can use print preview to check the resulting layout Shortcut Keys lt A1t gt F U Print Preview command Use this command to display the layout as it would appear when printed The main window will be replaced with a print preview window in which one or two pages will be displayed in their printed format The print pre view toolbar offers you options to view either one or two pages at a time move back and forth through the document zoom in and out of pages and initiate a print job Shortcut Keys lt A1t gt F V Print Setup command Use this command to select a printer and a printer connection This command presents a Print Setup dialog box where you specify
59. ciency of 60 These modules are thermoelec trically cooled for low dark count rate and deliver pre shaped TTL pulses They are the most common detect ors for applications where NIR sensitivity is important e g single molecule detection To achieve the spe Page 8 PicoQuant GmbH PicoHarp 300 software v 2 3 cified timing accuracy exact focusing into the center of the active area is necessary There may also be a shift and or broadening of the IRF dependent on count rate Principles Behind the TCSPC Electronics Conventional TCSPC systems consist of the following building blocks PC Bus Trigger 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 The principle of a classical CFD as found in PicoHarp models 1 0 is the comparison of the original detector signal with an amplified inverted and delayed version of itself The signal derived
60. clicking the axes opens the same dialog The dialog is non modal so it can remain permanently open The panel will remember its last position when closed When it is opened the next time it will be at that screen position again Data Cursor Button Opens the data cursor dialog box You can use this dialog to mark and retrieve the data values of individual histogram channels A pair of cross hairs will be provided for marking data points with the mouse or key board When the data cursor dialog is activated the cross hairs will appear in the histogram display area By clicking on or near a data point the current cross hair black will snap to that point At the same time a second cross hair grey will jump to the previously marked point The data cursor dialog then shows the cur rent count and time values for these points as well as the corresponding differences deltas The data curs or always applies to one active curve only You can select this active curve at the top of the dialog where it is shown with its corresponding curve colour While normally the grey cross hair jumps to the previous data point you can modify this behaviour by holding the lt SHIFT gt key down while clicking on curve points The grey cross hair will then remain at its previous position This allows finding a particular point while keeping the other one fixed Another advanced mode of operation of the data cursor is possible via the lt CurLeft gt and lt CurRight
61. collection of spectrally resolved lifetime histograms Data is collected as in stand ard Integration Mode and saved in different blocks of memory for each wavelength Routing is not supported in TRES mode lf a monochromator with appropriate stepper motor and associated drivers is installed a special ized dynamic link library Mono d11 can be installed together with the PicoHarp software The presence of this DLL will be detected by the software In this case the button for Monochromator and TRES control on the toolbar will be enabled Note that most of the monochromators can be m controlled only through dedicated stepper motor hardware from PicoQuant usually installed as part of a complete PicoQuant spectrometer Custom configurations can only be supported upon special request Clicking this button will launch a dialog for manual monochromator control and TRES setup There you can set up parameters such as the start step and end of the wavelength scan There will be error messages if the monochromator stepper and associated drivers are not configured properly Monochromator TRES Dialog The monochromator used for TRES measurements is controlled by a special dialog It shows a tab control and a status panel at the bottom of the dialog The three tabs give access to the nitialization TRES and Manual page of the dialog The status panel shows the current monochromator position in nm and whether the monochromator is cur rently moving t
62. con that you can click for details see also section 8 1 The rightmost area of the status bar indicates if the lt Tab gt and lt NumLock gt keys are latched down Axis Panel command Use this command to display the axis settings panel The result is the same as the axis panel button in the toolbar or double clicking the on the histogram axes The axis settings panel can be closed by clicking its close button Control Panel command Use this command to display the control panel This is the same as the control panel button in the toolbar The control panel can be closed by clicking its close button Page 49 PicoQuant GmbH PicoHarp 300 software v 2 3 Trace Mapping command Use this command to display the trace mapping dialog This is the same as the corresponding button in the toolbar The trace mapping dialog can be closed by clicking its close button 6 6 4 Help Menu The Help menu offers the following commands which provide you assistance with this application Help Topics Offers you the contents list of topics on which you can get help Help Index Offers you an index to topics on which you can get help Help Search Offers you a means of full text search on all help topics Activate Context Help Switches the cursor mode to point to item for context help About PicoHarp Displays version information of the PicoHarp software Note Online help context help on most functions dialogs control items e
63. cords 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 Page 33 PicoQuant GmbH PicoHarp 300 software v 2 3 5 5 TTTR Measurements with Real Time Correlation An important application of the PicoHarp 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 col lected This capability can be very useful in setting up and monitoring of FCS experiments The PicoHarp software provides such a real time software correlator for T2 and T3 mode It is available if the TTTR mode option has been purchased Although it collects the same T2 or T3 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 xi Correlation Corn Du DI axis Fit A B loer lirit or TT GO D jo DS i AA O175 17166 SR iM BB 0 178 17323 upper Int gt Wa AB 0 112 10977 E IT SS t aus sec Ai j
64. correlation curve Calculating more points is more time consuming and therefore it may lead to lower count rate limits that can be handled In this case you may get FIFO overruns The default of subsmp 8 is a reasonable tradeoff between speed and resolution On faster computers the setting of subsmpl 16 is a good choice Note that the starting point and spacing of the Tau sampling points also depends on the time tag resolution In T3 mode this corresponds to the sync period In T2 mode the native resolution of 4 ps is binned down to a time tag resolution of 25 ns in order to make the data manageable for the real time correlation algorithm The correlation curve display can be shown with a grid see software settings dialog accessible from the toolbar of the main window It also shows two useful figures obtained from the collected data top right of curve window The first figure is an approximation of G 0 In classic FCS experiments this corresponds to the inverse of the number of particles in the focal volume It is continuously updated together with the curve display which can be useful for system adjustment notably when using the repetitive accumulation mode Osc Note that the approximation is a simple averaging over the Tau points between 0 5 and 10 us The reason for not starting at O is that detector afterpulsing is often creating atefacts below 0 5 us The figure B is an indicator for molecular brightness It is also updated continuousl
65. ctory you selected for in Stallation and drag the icon onto the desktop This will create a link to the PicoHarp executable file You can now start it conveniently by double clicking the icon on the desktop You can also start up the PicoHarp software directly from a PicoHarp histogram file phd by double click ing on the file or dragging it onto the PicoHarp icon 4 2 The Main Window The PicoHarp software provides a measurement control interface to the PicoHarp hardware and an online histogram display The PicoHarp main window accommodates the histogram display area The histogram curves are displayed here 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 They 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 char
66. current value In this case changes take effect immediately without pressing lt Enter gt See Setting up the Input Channels to get started See also Input Channel 1 Discriminator Level Input Channel 1 Discriminator Level edit box and spin control Here the discriminator level of the Constant Fraction Discriminator CFD for the Channel 1 input can be set Units are millivolts mV the permitted range is 0 to 800 This corresponds to real input voltage levels of O to 800 mV Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the current value In this case changes take effect immediately without pressing lt Enter gt See Setting up the Input Channels to get started See also Input Channel 1 Zero Cross Resolution edit box and spin control Use this set of input controls to specify the acquisition resolution Units are picoseconds ps Possible choices are the base resolution 4 ps and binary multiples of this resolution 8 16 32 512 ps Type the desired resolution value ps as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the current value In this case changes take effect immediately without pressing lt Enter gt In case of entering values other than valid resolutions the next suitable resolu tion step is chosen automatically There are always 2 6553
67. curves in memory max 512 that have been filled by a measurement In addi tion to this all settings are memorized in the Windows registry so that at program startup you find the con trol panel as it was when you last closed the PicoHarp software Page 25 PicoQuant GmbH PicoHarp 300 software v 2 3 5 3 Routed Measurements 5 3 1 General Setup and Operation Classic TCSPC measurements are performed with one photon detector only However it is often desirable to have multiple detector channels in parallel without having to install multiple TCSPC electronics This can be achieved by detector routing into one set of TCSPC electronics The PicoHarp 300 but not the low cost model PicoHarp 300E supports routing of up to four detectors De tectors with TTL output usually Perkin Elmer SPCM AQR can be routed through the router module PHR 402 Up to four SPAD detectors with NIM output usually PDM series from Micro Photon Devices Italy can be routed through the PHR 403 router For ultimate flexiblity the universal router PHR 800 can be used PicoHarp hardware version 2 0 only It supports not only TTL and NIM but also any custom signal levels and polarities It can also be fitted with CFDs for use with PMT or MCP detectors All routers must be ordered as add on options The routing principle of the PHR routers is signal driven multiplexing It is also referred to as statistical time sharing Le the four router channels share
68. de lines stairs Prompt overwrite warning be fore overwriting existing data and TTTR Marker Edges The control connector of the PicoHarp 300 provides TTL inputs for synchronization signals The markers can be recorded at the rising or falling edge of the cor responding TTL signal The active edges can be chosen here The dialog also allows selective disabling of warnings that you do not wish to receive All settings will be kept in the Windows registry and will be re trieved at the next program start On multi user systems 2000 XP Vista they will be individual to each user 6 3 Control Panel Input Channel 0 Zero Cross edit box and spin control Here the zero cross level of the Constant Fraction Discriminator CFD for the Channel 0 sync input can be set Units are millivolts mV the permitted range is O to 20 Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the current value In this case changes take effect immediately without pressing lt Enter gt See section 5 1 Setting up the Input Channels to get started See also Input Channel 0 Discriminator Level Page 42 PicoQuant GmbH PicoHarp 300 software v 2 3 Input Channel 0 Discriminator Level edit box and spin control Here the discriminator level of the Constant Fraction Discriminator CFD for the Channel 0 sync input can be set Units are millivolts mV the permitted ran
69. dedicated cable for power supply and control pur poses connector RT CTRL Both cables must but be of approximately equal length max 2 m The PHR 40x routers must be connected to the RT connector through a 15 pin high density Sub D cable The PHR 800 can only be used with a PicoHarp model 2 0 and must be connected to the CTRL connector through a dedicated cable DVI I If you wish to use routing together with other devices marker input etc the other free control port can be used see section 8 3 2 on Router Control Port Connectors Alternatively a suitable Y cable for the simul taneous connection to the RT port can be used available from PicoQuant Note that this cable must not be extended Only required marker signals may be extended to connect the marker source The PHR 800 provides its own inputs for marker signals The following figure shows the connections required in a routing setup with four SPAD detectors and the PHR 402 403 For the PHR 800 the setup is nearly the same only that the CTRL port is used 1 Birch Holmes Imhof Nadolski and Cooper Multiplexed time correlated single photon counting SPIE Vol 909 Time Resolved Laser Spectroscopy in BioChemistry 1988 Page 26 PicoQuant GmbH PicoHarp 300 software v 2 3 sync from laser or sync detector max 2m PC max 2m PHR 402 3 out 1 2 3 4 SPAD SPAD ISPAD SPAD The Router will show its power state via a green LED and its trigger state si
70. during the run Also you can watch the status bar to see Page 38 PicoQuant GmbH PicoHarp 300 software v 2 3 the current wavelength and stepping activity Note that for a TRES measurement the control panel setting Stop OT applies as in Integration mode while Restart is meaningless in TRES mode TRES Data Analysis and Visualization Having collected a complete TRES data set you can save it to a regular phd file You can also inspect indi vidual curves via the Trace Mapping dialog You can furthermore use the software tool FluoPlot from Pi coQuant to visualize and analyze the data in various 2D and 3D representations with a multitude of options for colouring scaling and changing view aspects in 3D The figures below show FluoPlot visualizations from a TRES measurement of mixed oxazine dyes in ethanol counts time ns 600 0 65 7 wavelength nm V U 625 0 e ZZ 650 0 _ l i 675 0 101 5 ge 143 4 700 0 wem Nay W IW d it Ou Hi ui ID It nn 675 0 Mihi Mm JI d Ou Mira 650 0 E Lt wavelength nm pl 625 01 d d it i Wir j l Min li til Mil i d 600 i i i 65 7 77 6 29 5 101 5 113 4 s time ns FluoPlot is provided on your software installation disk s Just run FluoPlotSetup exe from there Note that the program requires modern hardware acceleration with support for OpenGL version 1 5 or higher Speedy handling of large data files requires suf
71. e PicoHarp Make sure that the host controller drivers are correctly installed otherwise they may be running only in USB 1 0 mode This may require using the mainboard manufacturer s driver disk rather than relying on Windows drivers alone Do not run other bandwidth demanding devices on the same USB interface when working with the PicoHarp T2 Mode In T2 mode both signal inputs of the PicoHarp 300 are functionally identical There is no dedication of input channel 0 to a sync signal Usually both inputs are used to connect photon detectors The events from both channels are recorded independently and treated equally In each case an event record is generated that contains information about the channel it came from and the arrival time of the event with respect to the overall measurement start The timing is recorded with 4 ps resolution Each T2 mode event record consists of 32 bits There are 4 bits for the channel number and 28 bits for the time tag If the time tag overflows a special overflow marker record is inserted in the data stream so that upon processing of the data stream a theoretically infinite time span can be recovered at full resolution Only for practical reasons of the current software design the number of records is limited to 2147483648 which corresponds to 8 GBytes of data Dead times exist only within each channel 90 ns typ but not across the channels Therefore cross correla tions can be calculated down to zero lag time
72. e acquisition time measurement time and the file for saving the data are the only parameters that can be set separately A typical approach to set up a TT TR mode measurement would be by first starting oscilloscope mode with an acquisition time of e g 1 second Then all control panel settings should be optimized to reliably obtain the expected data Once all settings are satisfactory click the TTTR Mode Button W on the toolbar This will bring up the TTTR Mode Dialog PicoHarp TTTR Mode E sl Acquisition Settings Note This mode performs real time data collection T2 Mode Do not run other programs to awad loss of data e Ve Mode Status Acq Time z M Wam overwrite Elapsed Time Ch Cnt Rate Chi CntAate Records bp 8 F Append numbers 00 00 00 1 00e 007 Z iet 0 stp detauilt pt3 E ready to start _stanT am CANCEL The dialog section Acquisition Settings is for the selection of the measurement mode T2 T3 an overall ac quisition time and a file name Note that switching between T2 and T3 mode takes some time because the hardware must be reconfigured Normally such a switching should not occur often because the two modes usually require a very different experiment setup The section Acquisition Settings also has two tick boxes for the handling of existing files You can turn on a warning and or automatically have numbers appended to the file name so that you can conveniently perform series of
73. e ambient temperature is 15 to 35 C Do not obstruct the cooling fan at the back and the air inlets at the bottom of the housing Page 41 PicoQuant GmbH PicoHarp 300 software v 2 3 A Trace Map Button Launches the trace mapping dialog The PicoHarp can measure histograms in up to 512 memory blocks Out of these up to 8 curves can be displayed The trace map dialog is used to select the curves to display Tick the individual show boxes to display a curve Select the number of the memory block you wish to map the individual display curve to Choose the active memory block you wish to use for the next measurement in the PicoHarp control panel UI ruj TTTR Mode Button disabled if this mode is not available Opens the TTTR mode dialog box Use this dialog to enter the acquisition time and the destination file for a TTTR mode run Make sure all other measurement parameters have been set and tested in interactive mode before entering TTTR mode Note TTTR mode is not part of the standard product and must be purchased separately This includes T2 and T3 Mode If the option is not installed the buton will remain disabled grey md TTTR Real Time Correlator Button disabled if TTTR mode is not available Opens the TTTR mode real time correlator dialog box Use this dialog for FCS preview during a T2 or T3 mode run Make sure all other measurement parameters have been set and tested in interactive mode be fore entering TTTR correlator mode C
74. e extent also be corrected for but this can be extremely difficult in practice For further details see our TechNote on TCSPC or the literature given in section 2 Warming Up and Calibration Observe the warm up period of at least 20 minutes dep on ambient temperature before using the PicoHarp for serious measurements Warming up takes about 20 to 30 minutes dependent on the lab temperature It is completed when the cooling fan starts to run for the first time You can use this time for set up and prelimin ary measurements Calibrate one more time before starting serious measurements The calibration takes only a few seconds For very long measurements allow some more time for thermal stabilization and avoid temperature changes gt 5 K in the environment of the instrument The permissible ambient temperature is 15 to 35 C Do not obstruct the cooling fan at the back and the air inlets at the bottom of the housing Make sure that the cooling air can circulate freely and no other hot instrument is under the cooling inlets Custom Programming of the PicoHarp A programmer s library DLL for custom Windows software development is available to build your own ap plications e g in LabVIEW C Delphi Lazarus Matlab or Visual Basic A rich set of demo code is provided for an easy start A Library for Linux is also available It provides the same interface so that applic ations can easily be ported between the platforms Software Updates We c
75. e to a specified file name Print Prints the currently displayed histogram Page Setup Allows modifying the page layout for printing Print Preview Displays the layout as it would appear printed Print Setup Selects a printer and printer connection 1 4 lt Recent Filename gt Opens one of the four last recently opened files Exit Exits PicoHarp software New command Use this command to create a blank histogram with the last default settings You can open an existing histogram file with the Open command Shortcuts Toolbar Keys lt Ctri gt N lt ALt gt F N Page 46 PicoQuant GmbH PicoHarp 300 software v 2 3 Open command Use this command to open an existing histogram file All control panel settings will be restored also You can also open PicoHarp data files phd by double clicking them or dragging them onto the PicoHarp icon You can revert to a blank histogram and default control panel settings with the New command You can also select other file types for loading as histogram data notably T3 Mode files Loading such files as histogram data is for diagnostics only and cannot use their full information content Shortcuts Toolbar Keys See lt Aleere File Open dialog box The following options allow you to specify which file to open Look in Select i e browse into the directory in which the file that you want to open resides Main box In this box you see the content of the
76. ectors will go into the active block selected in the control panel Note that in this case the router settings in the curve details display are partially marked n a because they are no longer directly mappable to one input Page 27 PicoQuant GmbH PicoHarp 300 software v 2 3 TTTR mode measurements see section 5 4 on Time Tagged Mode Measurements always implicitly provide routing information if a router is used The routing information from the PHR 40x is represented by two bits in each TTTR record allowing the distinction of up to four routing channels Routing does not affect TT TR throughput in any way other than given by the limits in total count rate 9 3 2 Using the PHR 800 Router The PHR 800 allows very fine control of its input settings For this purpose the PicoHarp software provides a dedicated dialog It can be launched through the PHR 800 button on the Toolbar The button will remain disabled grey if no PHR 800 was detected at startup The control dialog for the PHR 800 is shown below It has four sections for the input configuration and four section for CFDs The router is modular and can be purchased without the full set of CFDs In this case the corresponding dialog sections are not accessible greyed out PHR 800 Settings E x C Custom Custom C Custom Level mi Level mid Level mid Level mi 200 200 200 H 200 H Campe Be Be Campe f Falling f Falling f Falling Ge Falling LED
77. el 0 CFD discriminator level in mV Brd i CFDZeroCross1 int32 input channel 1 CFD zero cross level in mV Brd i CFDLevel1 int32 input channel 1 CFD discriminator level in mV Brd i Resolution float resolution in ns Brd i RouterModelCode int32 O none 1 PHR_402 2 PHR_403 3 PHR_800 Brd i RouterEnabled int32 1 enabled O disabled routing off The following block will appear in the file for each j 1 to lt NumberOfRouterChannels gt currently this must be 4 Brd i RtCh j InputType int32 O custom 1 NIM 2 TTL Brd i RtCh j nputLevel int32 in mV Brd i RtCh j InputEdge int32 1 rising O falling Brd i RtCh j CFDPresent int32 O not present 1 present Brd i RtCh j CFDLevel int32 in mV Brd i RtCh j CFDZCross int32 in mV end of block The following is a T2 T3 Mode specific header ExtDevices int32 external devices bitwise coded Reserved int32 reserved Reserved int32 reserved InpRate0 int32 input O count rate as displayed by rate meter InpRate1 int32 input 1 count rate as displayed by rate meter StopAfter int32 stopped after this many ms StopReason int32 O timeover 1 manual 2 overflow NumRecords int32 number of T3 event records ImgHdrSize int32 size of special header to follow before the records start end of block The following is a special header that may not be present at all for each i 0 to lt ImgHdrSize gt Reserved i uint32 reserved end of block
78. enu offers the following commands Toolbar Shows or hides the toolbar Status Bar Shows or hides the status bar Axis Panel Shows the axis settings panel Control Panel Shows the control panel Trace Mapping Shows the trace mapping dialoq Toolbar command Use this command to display and hide the toolbar which includes buttons for some of the most common commands such as File Open A check mark appears next to the menu item when the toolbar is displayed Status Bar command Use this command to display and hide the Status Bar which describes the action to be executed by the se lected menu item or depressed toolbar button the current measurement activity and keyboard latch state A check mark appears next to the menu item when the Status Bar is displayed The status bar is displayed at the bottom of the PicoHarp main window To display or hide the status bar use the Status Bar command in the View menu The left area of the status bar describes actions of menu items as you navigate through menus This area similarly shows messages that describe the actions of toolbar buttons as you depress them before releasing them If after viewing the description of the toolbar button command you do not wish to execute the command then release the mouse button while the mouse pointer is not over the toolbar but ton The second area from the left shows the current measurement status of the PicoHarp Next to it you may find a warnings i
79. ere R is the chosen resolution At the highest possible resolution of 4 ps this results in a span of 16 ns If the time difference between photon and the last sync event is larger the photon event cannot be recorded This is the same as in histogramming mode where the number of bins is also finite However by choosing a suitable sync rate and a compatible resolution R it should be possible to reason ably accommodate all relevant experiment scenarios R can be chosen in doubling steps between 4 ps and 512 ps Dead time in T3 mode is the same as in the other modes 90 ns typ Since there is only one photon chan nel only autocorrelations can be calculated unless a router is used This can be done at the resolution R but of course only starting from lag times larger than the dead time The 32 bit event records are queued in a FIFO First In First Out buffer capable of holding up to 256 k event records The FIFO input is fast enough to accept records at the full speed of the time to digital converters up to 10 Mcps each This means even during a fast burst no events will be dropped except those lost in th dead time anyhow The FIFO output is continuously read by the host PC thereby making room for fresh in coming events Even if the average read rate of the host PC is limited bursts with much higher rate can be recorded for some time Only if the average count rate over a long period of time exceeds the readout speed of the PC a FIFO overrun co
80. erform a numerical expo nential 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 r m s 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 pack age from PicoQuant provides this functionality Further Reading 1 Lakowicz J R Principles of Fluorescence Spectroscopy 3 Edition Springer Science Business Media New York 2006 2 O Connor D V O Phillips D Time correlated Single Photon Counting Academic Press London 1984 3 O Connor D V O Ware W R Andre J C Deconvolution of fluorescence decay curves A critical comparison of techniques Journal of Physical Chemistry 83 1333 1343 1979 4 Becker W Advanced Time Correlated Single Photon Counting Techniques Springer Berlin 2005 5 Wahl M Rahn H J Gregor l Erdmann R Enderlein J Dead time optimized time correlated photon counting instrument with synchronized independent timing channels Review of Scientific Instruments Vol 78 033106 2007 D http www picoquant com _biblio htm Bibliography listing all publications with work based on PicoQuant instruments 7 http www picoquant com showpapers php sid Bibliography of publications of wor
81. ess of your measurement in the status boxes The leftmost status box shows the elapsed measure ment time The two boxes in the center show the count rates The rightmost box will show the number of events that have been recorded Note that this includes overflow and marker records Therefore the number of records will be somewhat larger than the true photon counts The overflow and marker records will be re moved by the data analysis processing software Page 31 PicoQuant GmbH PicoHarp 300 software v 2 3 A measurement can be stopped at any time by clicking the Stop button the middle button The data recor ded up to this point will be in the file When the measurement has completed the Stop button will change to grey disabled Use the Cancel button to return to the normal interactive mode Again this will take some time for hardware reconfiguration External Markers Often it is desirable to synchronize TCSPC measurements with other information or processes of complex measurement tasks In order to perform e g Fluorescence Lifetime Imaging the spatial origin of the photons must be recorded as well For this purpose one needs a mechanism to assign external synchronization in formation to the TCSPC data For the special case of Fluorescence Lifetime Imaging conventional systems use large arrays of on board memory and switch to new blocks of memory upon arrival of e g a pixel clock pulse To accommodate the large amount of data ge
82. et to None Similarly the divider must not be used if the channel 0 input is used for a photon detector coincidence correlation measurements etc In summary The PicoHarp 300 is designed to always work in forward start stop mode Experimental Setup for Fluorescence Decay Measurements with TCSPC The figure below shows a typical setup for fluorescence lifetime measurements with TCSPC The pico second 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 lt 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 excita tion light by means of an optical cutoff filter other configurations may utilize a monochromator here Then it Page 10 PicoQuant GmbH PicoHarp 300 software v 2 3 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 H5783 PMT from Hamamatsu is very convenient It only needs a 12 V supply
83. fications and external appearance are subject to change without no tice Common Specifications Input Channels Trigger principle Trigger point Input voltage operating range pulse peak 50 Q optimum Input voltage max range damage level CFD level adjust CFD zero cross adjust Trigger pulse width Trigger pulse rise fall time Max count rate Max sync rate without divider Max sync rate with divider 1 8 Time to Digital Converter Base resolution min time bin width Timing uncertainty rms Time base calibration error Differential non linearity full measurement range Dead time Max conversion rate Histogrammer Maximum number of time bins Bin widths Measurement ranges Acquisition time TTTR Engine T2 mode resolution T3 mode resolution FIFO buffer depth records Sustained throughput sum of channels Acquisition time Page 64 Constant Fraction Discriminator Falling edge O mV 1 000 mV 200 mV 400 mV 2 000 mV 800 mV 20 mV 20 5 ns lt 2ns 10 0 10 cps 10 0 MHz 84 0 MHz OmV OmV 4 ps lt 12 ps 1078 at lt 50 ppm lt 5 0 p p lt 1 0 rms lt 95 ns 10 0 MHz 65 536 4 8 16 512 ps 65 536 4 ps 65 536 512 ps 1 ms 100 h 4 ps 4 8 16 512 ps 262 144 typ 5 10 events s 1ms 100h PicoQuant GmbH PicoHarp 300 software v 2 3 Operating Enviroments Ambient temperature ee eres is 6 59 F 9
84. ficient memory Further data analysis may need to be performed by dedicated software either custom programs or specialized solutions available from PicoQuant Page 39 PicoQuant GmbH PicoHarp 300 software v 2 3 6 Controls and Commands Reference 6 1 Main Window The Title Bar The title bar is located along the top of a window To move the window drag the title bar Note You can also move dialog boxes by dragging their title bars The title bar may contain the following elements System Menu iil lt Filename gt PicoHarp Minimize Button B Maximize or Restore Button resp mj eil Close Button x Scroll bars Displayed at the right and bottom edges of the PicoHarp window if a certain minimum window size is reached The scroll boxes inside the scroll bars indicate your vertical and horizontal location in the display area You can use the mouse to scroll to other parts of the window Size command Use this command to resize the active window Note The command is unavailable for already maximized or minimized windows Shortcut Mouse Drag the corners or edges of the window Keys lt Alt gt lt Space gt S Minimize command Use this command to reduce the PicoHarp window to an icon Running measurements will continue Note The command is unavailable for already minimized windows Shortcut Mouse Click the minimize button on the title bar Keys lt Alt gt lt Space gt N Maximize command
85. forma A tion about the input signals and the current acquisition settings If these settings together with the input rates indicate possible errors the software will activate the warning icon The warning icon can be clicked to display a list of current warnings together with a brief explanation of each warn ing see also section 8 1 The rightmost area of the status bar indicates if the lt Tab gt and lt NumLock gt keys are latched down Page 17 PicoQuant GmbH PicoHarp 300 software v 2 3 4 3 The Toolbar The toolbar is displayed across the top of the PicoHarp main window below the menu bar The toolbar provides quick mouse access to frequently used commands and tools Note that some buttons may be grayed out disabled dependent on the installed software components and or the state of the hardware Deg ml ai ze LAH wf le Ein vol 3 mg To hide or display the Toolbar choose Toolbar from the View menu lt ALT gt V T The toolbar is capable of docking Le you can move it to another edge of your window or even have it displayed as a separate win dow The following table explains the individual buttons Click To Open a blank histogram with default control panel settings Open an existing histogram file Displays the Open dialog box in which you can locate and open the desired file Save the current histogram data with its current name If you have not named the file the Save As dialog box is displayed
86. ft rate meter in the Pico Harp main window Note that the rate meter internally corrects for the chosen divider setting so that the meter always shows the undivided input rate The meter display should therefore match the rate delivered from the source The rate meter will be refreshed every 0 1 to 1 seconds determined by the value in the Software Settings Dialog The Input 0 sync rate should be displayed very accurately Large fluctuations or occasional zeros indicate an incorrect discriminator level setting or an unstable sync signal Try to vary the discriminator level to obtain a stable rate meter reading If the sync rate is stable and as expected you can proceed to the set up of the other input A last fine tuning of the discriminator and zero cross level can be done when the detector input is up and running Setting Up the Channel 1 Input As noted before the PicoHarp 300 input channels are designed electrically identical both with a CFD They can handle an input voltage range of 0 to 1 V TTL SPAD detectors Perkin Elmer SPCM AQR deliver positive pulses of 3 V and must therefore be connected through a pulse inverter with attenuation Pi coQuant SIA 400 Note that this is not required when using a router PMTs should be connected through a Page 22 PicoQuant GmbH PicoHarp 300 software v 2 3 preamplifier available from PicoQuant MCP PMT detectors should be connected through an amplifier with slightly higher gain Be sure
87. ftware v 2 3 6 5 Trace Mapping Dialog The PicoHarp software can measure histograms in up to 512 memory blocks Out of these up to 8 curves can be displayed The trace mapping dialog is used to select the curves to display Tick the individual boxes Show to display a curve Select the number of the memory block you wish to map the individual display curve to The Trace Mapping Dialog also provides some statistics on each curve These items are e FWHM The Full Width Half Maximum of the curve peak usually for IRF traces e Max Count The count in the highest point of the curve e At Time The time corresponding to the Max Count channel e Resolution The channel width bin width of the curve Furthermore there are several buttons e Details Can be clicked to see more curve information e All Can be clicked to tick all traces as shown e 0 7 Can be clicked to set the default mapping of trace 0 7 to block 0 7 e Clear trash can Can be clicked to delete the contents of individual blocks The trace mapping dialog can be launched from the corresponding button on the toolbar as well as through the trace colour indicator on the control panel 6 6 Menus 6 6 1 File Menu The File menu offers the following commands New Opens a new data file clears all histogram data and restores default settings Open Opens an existing histogram file Save Saves a histogram file Save AS Saves an opened histogram fil
88. ge is 0 to 800 This corresponds to real input voltage levels of 0 to 800 mV Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the current value In this case changes take effect immediately without pressing lt Enter gt See Setting up the Input channels to get started See also Input Channel 0 Zero Cross Input Channel 0 Sync Divider selection box Here the programmable divider of the sync input can be set This allows to reduce the sync input rate so that the period is at least as long as the dead time This is required for fast sync sources gt 10 MHz Internal lo gic 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 With random and low rate signals lt 1 MHz e g in coincidence correlation experiments the divider must be set to None Input Channel 1 Zero Cross edit box and spin control Here the zero cross level of the Constant Fraction Discriminator CFD for the Channel 1 input can be set Units are millivolts mV the permitted range is O to 20 Type the value as an integer in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the
89. gnals at any input via a yellow LED If the router is connected the PicoHarp software will detect its presence at start time In this case the Routed check box _ Routed in the control panel will be active otherwise disabled and grey F Routed When using a PHR router the PicoHarp s CFD level in the control panel should be set between 50 and 350 mV The precise figures should be optimized experimentally The PHR 40x routers are configured for fixed input signals TTL or NIM respectively The PHR 800 allows adjustment of input level and polarity See the corresponding section 5 3 2 on Using the PHR 800 Router further below If a router is correctly connected you can switch to routed data acquisition by ticking the I Routed check box In routed measurements the counts from each detector are collected in different blocks of on board memory thereby forming one histogram for each detector This can be used e g to collect different polariza tion directions or colours at the same time Note that the router acts as a multiplexer so that the operation is only quasi parallel In particular the four channels must share the channel 1 input of the PicoHarp 300 so that the total count rate cannot exceed that permitted by the PicoHarp channel When routing is enabled I Routed the collection of histograms will operate on four consecutive blocks of memory simultaneously Recall that there are 512 blocks of Tre Block memory in total In ro
90. gt ar rows of the keyboard Pressing these keys will direct the black cursor to the next left or right point in the curve Again the behaviour of the marker for the previous point grey cross can be controlled via the lt SHIFT gt key Control Panel Button Launches the PicoHarp control panel If the control panel is already open subsequent clicking of this button will just make the control panel the active window The dialog is non modal so it can remain permanently open The panel will remember its last position when closed When it is opened the next time it will be at that screen position again even in the next PicoHarp session Calibrate Button Calibrates the time measurement circuits This is necessary whenever temperature changes gt 5 K occurred Calibrate after warming up of the instrument before starting serious measurements Warming up takes about 20 to 30 minutes dependent on the lab temperature It is completed when the cooling fan starts to run for the first time Calibration takes only a few seconds During calibration the status bar shows the message CALIB RATING Calibration is only important for serious measurements You can use the instrument during the warming up period for set up and preliminary measurements For very long measurements allow some more time for thermal stabilization calibrate immediately before the measurement and try to maintain a stable room temperature during the measurement The permissibl
91. hanging to this mode takes a few seconds for reconfiguration of the hardware Note TTTR mode is not part of the standard product and must be purchased separately See section 5 6 on Time Resolved Emission Spectra gt PHR 800 Router Setup Button lf a PHR 800 router is connected to the PicoHarp this button will be enabled It launches a dialog for setting up the inputs and the built in CFDs for the four router channels The settings will be stored in the Windows registry and the dialog does not need to be used again once satisfactory settings for a given measurement setup have been established Note that the settings are also retrieved upon loading a data file Monochromator TRES Button disabled if device is not available In order to measure Time Resolved Emission Spectra the PicoHarp software provides a TRES measure ment mode that allows to control a monochromator via stepper motors and automated collection of spec trally resolved lifetime histograms Clicking this button will launch the dialog for manual monochromator con trol and TRES setup There you can set up parameters such as the start step and end of the wavelength scan There will be appropriate error messages if the monochromator stepper and associated drivers are not configured properly Software Settings Button Opens the software settings dialog box Use this dialog to change standard settings of the PicoHarp soft ware Notably these are Display rate 0 1 to 1s Draw mo
92. har 20 reserved for automated measurements The following block will appear in the file for each i 1 to lt NumberOfBoards gt currently this must be 1 Brd i Hardwareldent char 16 currently PicoHarp 300 Brd i dardwareVersion char 8 currently 1 0 or 2 0 Brd i HardwareSerial int32 hardware serial number Brd i SyncDivider int32 input channel 0 Sync input divider Brd i CFDZeroCross0 int32 input channel 0 CFD zero cross level in mV Brd i CFDLevel0 int32 input channel 0 CFD discriminator level in mV Brd i CFDZeroCross1 int32 input channel 1 CFD zero cross level in mV Brd i CFDLevel1 int32 input channel 1 CFD discriminator level in mV Brd i Resolution float resolution in ns Brd i RouterModelCode int32 O none 1 PHR_402 2 PHR_403 3 PHR_800 Brd i RouterEnabled int32 1 enabled O disabled routing off The following block will appear in the file for each j 1 to lt NumberOfRouterChannels gt _ currently this must be 4 Brd i RtCh j InputT ype int32 O custom 1 NIM 2 TTL Brd i RtCh j InputLevel int32 in mV Brd i RtCh j InputEdge int32 1 rising O falling Brd i RtCh j CFDPresent int32 O not present 1 present Brd i RtCh j CFDLevel int32 in mV Brd i RtCh j CFDZCross int32 in mV end of block end of block The following block will appear in the file for each i 1 to lt NumberOfCurves gt
93. hat 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 re gister only one photon per excitation emission cycle If now the number of photons occurring in one excita tion cycle were typically gt 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
94. he red LED labelled Stepping is flashing when it moves EE Stepping 737 00 nm The dialog is non modal i e it does not need to be closed before other windows can be accessed It has its own icon in the Windows Taskbar from where it can be brought back to the top should it have been covered by other windows Monochromator Models The type of the current monochromator is displayed in the title bar of the dialog Apart from a simulation mode as of this release the following monochromator models are supported Sciencetech ST 9030 ST 9030DS and ST 9055 Acton Research SP 2150 SP 2155 SP 150 and SP 275 Some other Acton Re search models are partially supported Note that some monochromators can only be controlled through ded icated stepper motor hardware from PicoQuant or the monochromator supplier Usually these are installed as part of a complete PicoQuant spectrometer Please contact PicoQuant for details If your system is not configured for use of a monochromator the Monochromator TRES dialog shows the caption Simulation This is for demonstration purposes only It will work the same way as the real ST 9030 monochromator described below except that pressing the nitialize button you will be prompted to decide whether the initialization should be successful or not To continue with the demonstration press Yes Of course the simulated monochromator movements will not have any effect on the measured TCSPC h
95. his case changes take effect immediately without pressing lt Enter gt The default increment decrement per mouse click is logarithmic Trc Block trace colour indicator edit box and spin control The PicoHarp can measure histograms in up to 512 memory blocks Out of these up to 8 curves can be dis played and one active block can be used for measurements Choose the active memory block you wish to use for the next measurement here The trace mapping dialog is used to select the curves for display Make sure the curve you are using is switched on shown You can open this dialog directly by clicking on the trace colour indicator The colour indicator shows the trace colour the chosen block is currently mapped to If it is a solid square the curve is mapped and shown If it is mapped but not shown the indicator shows a small striped square If the curve is not even mapped for display the indicator remains white Make sure not to overwrite existing data in a block that was used before In order to warn you if a trace is used the heading of the Trc Block selector will turn red Routed check box Enable Disable Routing The check box will remain disabled if no router is found at startup Restart check box If this box is ticked checked the measurement will automatically restart after the acquisition time has elapsed Toggle the current setting with a mouse click The setting is without effect in TRES mode Stop on Overflow check box
96. his 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 r m s error standard deviation or the Full Width Half Maximum FWHM of the timing distribution or instrument re sponse function IRF Note that these two notations are related but not identical Good but also very ex pensive detectors notably Micro Channel Plate PMTs can achieve timing uncertainties as small as 30 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 neces sary to obtain an electrical timing reference signal sync for comparison with the fluorescence photon signal Where this signal is derived from 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 nar row negative pulse of 800 mV into 50 NIM standard The sharp falling edge is synchronous with the laser pulse e g lt 3 ps r m s 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 lig
97. ht 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 electric 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 elec tronics to the total timing error is usually small For the PicoHarp it is typically lt 10 ps Finally it is always a good idea to keep electrical noise pickup 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 width strongly depends on their relative magnitude Strictly the overall IRF is the convolution of all com ponent 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 r m s figure according to statistical error propagation laws dE 2 system o
98. ified 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 Pi coQuant GmbH PicoHarp 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 ex planation and to the owner s benefit without intent to infringe 1 Trademarks of Microsoft Inc Page 13 PicoQuant GmbH PicoHarp 300 software v 2 3 3 2 Software Installation Before installing the new software you should if applicable back up all PicoHarp data files you created in any older PicoHarp directory previous versions etc as well as any critical data on your PC For a clean start we also recommend to uninstall all older PicoHarp software The PicoHarp 300 setup files are normally provided on a CD supplied with your PicoHarp device The in staller program file containing the complete distribution is setup exe If you received the software package by email 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
99. indows versions you will need administrator status to perform the setup 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 with the Windows Explorer If you unpacked the setup file to a hard disk Sen 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 PicoHarp files will be installed To avoid confu sion make sure not to specify the path of an older PicoHarp version that you have not uninstalled or that of any other program on your PC The default location is Program Files PicoQuant PicoHarp300v23 Note that dependent on your Windows installation the folder Program Files may be spelled in you na tional language Setup will also create a dedicated program folder for the new PicoHarp 300 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 PicoHarp version that you have not uninstalled or the dedicated folder of any other program
100. is warning is issued in histogramming and T3 mode when the rate at input 1 is over 5 of the rate at input 0 This is the d d classical pile up criterion It will lead to noticeable dead time artefacts There are rare measurement scenarios where this condition is expected and the warning can be disabled Examples are antibunching measurements WARNING_DIVIDER_GREATER_ONE You have selected T2 mode and the sync divider is set larger than 1 This is probably not intended The sync divider is designed primarily for high sync rates from lasers and requires a fixed pulse rate at channel 0 In that case you d should use T3 mode If the signal at channel O is from a photon detector coincidence correlation etc a divider gt 1 will lead to unexpected results There are rare measurement scenarios where this condition is intentional and the warning can be disabled WARNING TIME SPAN TOO SMALL This warning is issued in histogramming and T3 mode when the sync period 1 Rate chO is longer that the start to stop time span that can be covered by the histogram or by the T3 mode records You can calculate this time span as follows d d Histogramming mode Span Resolution 65536 T3 mode Span Resolution 4096 Events outside this span will not be recorded There are some measurement scenarios where this condition is intentional and the warning can be disabled WARNING_OFFSET_UNNECESSARY This warning is issued in histogramming and T3 mode
101. ise See sec tion 8 3 on Hardware Technical Data for pin assignments It is recommended to start instrument setup without anything connected to the control ports After connecting the PicoHarp to the PC via USB Windows should detect the device and install the driver Note that the software setup for the PicoHarp 300 pre installs the driver so that it should be found automatic ally When prompted for the driver location please point the driver installation wizard to the folder DRIVER in the installation folder or the equivalent folder on your installation media If you downloaded and unzipped the software to a temporary disk location please select the driver folder in that location 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 PicoHarp 300 as a USB device in the Windows device manager For troubleshooting see the section 3 4 Page 15 PicoQuant GmbH PicoHarp 300 software v 2 3 3 4 Installation Troubleshooting After completion of the software setup the PicoHarp device should be listed in the Windows Device Man ager under the USB tree Right click My Computer gt left click Properties gt 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 PicoHarp 300 and inspect its Properties You can also use the Windows system information facilities S
102. isto grams Page 36 PicoQuant GmbH PicoHarp 300 software v 2 3 Monochromator Initialization Before use the monochromator must be initialized This means it has to seek its mechanical reference posi tion determined by a micro switch Physical calibration values are kept in a monochromator configuration file monochromator cfg which is generated by the system supplier at the time of delivery Do not edit this file unless you have exact instructions for doing so Position initialization is required each time the software is started It can be controlled on the Initialization page of the dialog At first startup the dialog shows the state not initialized This means the software has no information about the current monochromator stepper position JE Mono ST 9030 E x Initialize not initialized The position initialization is started by the nitialize button If the initialization is running the caption of the button will change to Stop and the LED will start flashing You should also hear the stepper motor If the ini tialization is successful the string not initialized will change to Initialization OK otherwise it will change to Initialization failed In this case check cabling and power supply of the system This may happen if either the Stop button was pressed or an error occurred during the initialization process The Manual and TRES pages will not be available unless the initialization was successfully pe
103. k using the PicoHarp 300 Page 12 PicoQuant GmbH PicoHarp 300 software v 2 3 3 Hardware and Software Installation 3 1 General Installation Notes Read This First The PicoHarp software version 2 3 is compatible with Windows 2000 XP and Vista When handling the PicoHarp 300 system be sure to avoid electrostatic discharges especially when con necting cables In order to use the PicoHarp software with the PicoHarp 300 hardware a compatible device driver must be installed The PicoHarp 300 is a USB 2 0 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 prompt the user for the appropriate driver disk Note that the software setup for the PicoHarp 300 pre installs the driver in the application directory in a subfolder driver so that windows will find it there This requires that the software setup is performed before connecting the device In case you are prompted for the driver location please point the driver installation wizard to that folder Just in case the driver folder is also provided on the installation media Some windows versions verbosely warn about drivers not being validated by Microsoft You can ignore these warnings On some Windows versions you will need administrator status to perform software setup and deinstallation You can install the software for use by all use
104. lling the software Page 16 PicoQuant GmbH PicoHarp 300 software v 2 3 4 Software Overview 4 1 Starting the Program After correct installation the Start Menu Programs contains a shortcut to the PicoHarp software To start the PicoHarp software select PicoQuant PicoHarp 300 v2 3 Note that after switching the PicoHarp on you need to allow a warm up period of at least 20 minutes before using the instrument for serious measure ments You can use this time for set up and preliminary measurements If the PicoHarp 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 re main disabled This allows you to use the software without the PicoHarp hardware e g to view or print files on another computer If the PicoHarp 300 is correctly installed and there are still hardware related errors you can use the Win dows device manager for troubleshooting see section 3 4 If problems cannot be resolved see section 7 on Problems Tips amp Tricks for support If possible try the device on another computer For regular use of the PicoHarp you may want to create an icon for it on your Windows desktop You can do this conveniently via a right mouse click on the start menu entry Send To gt Desktop create shortcut Al ternatively use the Windows Explorer to locate the file PicoHarp exe in the dire
105. mand to display the copyright notice and version number of your PicoHarp software and hard ware if installed Shortcut Keys lt Alt gt H A Page 50 PicoQuant GmbH PicoHarp 300 software v 2 3 6 7 Tithe and Comment Editor You can use this dialog to edit the file title comment It can be opened via double click on the title via the File menu or via the Print Preview Toolbar The text you enter here will be stored in the data file The first line will be displayed as the file title over the histogram display area The text you can enter here is limited to 4 lines and 255 characters Upon loading of a data file the title and comment will also be retrieved It will also be included in prints if the corresponding check mark is set in the page setup dialog box available through the File menu 6 8 Print Preview Dialog The Print Preview command File menu opens the Print Preview Dialog Use this Dialog to preview the lay out as it would appear when printed When you choose this command the main window will be replaced with a print preview window in which one or two pages will be displayed in their printed format The print preview toolbar offers you options to view either one or two pages at a time move back and forth through the document zoom in and out of pages and initiate a print job It also offers a Page Setup button that opens a dialog for the print layout and editing the file comment Print Preview Dialog Toolbar The prin
106. 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 PicoHarp in all modern programming environments For details on the Time Correlated Single Photon Counting method please read section 2 as well as our TechNote on TCSPC and consult the literature referenced in Further Reading at the end of the next sec tion Experienced users of the method should be able to work with the PicoHarp straight away Nevertheless we recommend carefully reading the section 3 on Hardware and Software Installation to avoid damage Later the comprehensive help function of the PicoHarp software will probably let the manual gather dust on the shelf Page 4 PicoQuant GmbH PicoHarp 300 software v 2 3 2 Primer on Time Correlated Single 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 fa
107. measurements The file name is shown in red if the file already exists The button with the file icon will open a standard Windows file dialog You can select an existing file or choose a new name The Pico Harp TTTR mode files have the extension pt2 or pt3 respectively For maximum count rate through put you should choose a file destination on a fast local hard disk as outlined above Network drives are often too slow The dialog section Status shows elapsed time the count rates and the number of collected records Note that this is limited to 2147483648 records 8 GBytes of data Below these figures there is a status line show ing what is currently happening Further below there are buttons for Start Stop and Cancel Start and Stop control the actual TTTR measurement run Cancel is for leaving the TTTR mode dialog When the TTTR mode dialog is running it continuously collects information about the input sig nals and the current acquisition settings If these settings together with the input rates indicate possible errors the software will activate the warning icon The warning icon can be clicked to AN display a list of current warnings together with a brief explanation of each warning see also sec tion 8 1 In order to fix wrong settings you may have to close the TTTR mode dialog and return to the control panel The TTTR mode measurement will start as soon as you click the Start button You will then be able to watch the progr
108. 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 mo lecules of interest from background or other species This has made the technique very interesting for sens itive analysis even down to the single molecule level Of course other aspects than lifetime of the temporal pattern of the fluorescence decay are of interest too 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 relat ive 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 PicoHarp it is in no way restricted to this task Other important applications are e g Quantum Cryptography QC Time Of Flight TOF and Optical Time Domain Reflectometry OTDR as well as general coincidence correlation The PicoHarp 300 is a comp
109. mpatible synchronization signals that will be recorded as markers in TTTR mode The pins are internally pulled down so that they are inactive when left unconnected The active edge is chosen in the software settings dialog Rise fall times must be 10 ns or faster Both high and low state must be at least 100 ns long The clock period may therefore in principle be as short as 200 ns but data bus throughput constraints will apply Each marker creates an additional TTTR record so that one must en sure not to swamp the data stream with too many marker records Markers also take precedence over photon records so that excess marker traffic can suppress photon records In fast imaging applications it is therefore recommended not to use a pixel clock but a line clock only Because each photon has a time tag it is usually not necessary to use an additional pixel clock Note that the same signals can also be fed in via the CIR connector so that they can be used even if a router is connected Pins 11 provides a 5 V DC supply voltage external electronics can use Under no circumstances must this line be shorted or loaded in excess of the specified maximum current Note that the maximum current must be shared with any loads connected at the 5V line of the CTRL connector Page 66 PicoQuant GmbH PicoHarp 300 software v 2 3 Pins 12 and 14 are the common ground for the TTL signals and the DC supply line They are also connected to the chassis All othe
110. n involves the following steps first the time difference between the photon event and the corres ponding excitation pulse must be measured For this purpose both signals are converted to electric 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 Obvi ously 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 digit al 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 import ance Count Rates and Single Photon Statistics lt was already mentioned that it is necessary to maintain a low probability of registering more than one photon per cycle This was to guarantee t
111. n principle of course any kind of background activity is allowed Should the system become overloaded the PicoHarp histograms will not be wrong or distorted but measurement times and display rates may become irregular You can even iconize the PicoHarp during measurements without doing damage This may be of interest for lengthy measurements in integration mode where one is only interested in accumulating a certain amount of counts without need for permanent monitoring The panel meters showing the current count rates are not meant to be 100 accurate They merely serve as an aid for setting up the system Some of them may suffer from system overload Accordingly the values shown in the curve details trace mapping dialog are subject to such tolerances In TTTR mode system overload will manifest itself in loss of data Here no other background processing should be allowed The faster the PC the less these issues will matter TTTR mode performance i e max throughput without loss may also be improved by defragmenting the hard disk before long measurements The real time correlation is very demanding in terms of CPU load The correlator is implemented in a way that will make use of multiple processor cores It is therefore beneficial to use one of the recent dual or quad core CPUs USB Interface In order to deliver maximum throughput the PicoHarp 300 uses state of the art USB 2 0 bulk transfers This is why the PicoHarp must rely on ha
112. nal data file If the file is too large for email gt 10 MB please provide access to it on a public download server If you have serious problems that require the device to be sent in for inspection repair please request an RMA number before shipping the hardware Observe precautions against static discharge under all circum stances in handling packaging and shipping Use original or equally protective packaging material Of course we also appreciate good news If you have obtained exciting results with one of our systems please let us know and where appropriate please mention the instrument in your publications At our Web site we maintain a large bibliography of publications related to our instruments It may serve as a reference for you and other potential users See http www picoquant com _biblio htm Please submit your publica tions for addition to this list Page 53 PicoQuant GmbH PicoHarp 300 software v 2 3 8 Appendix 8 1 Warnings When the PicoHarp software is running with functional hardware it continuously collects informa tion about the input signals and the current acquisition settings If these settings together with the AN input rates indicate possible errors the software will indicate this by means of a warning icon While the software is running in interactive histogramming mode the warning icon is displayed at the bottom of the main window in the status bar In the case of TTTR mode it will appear directly
113. nerated due to the 3 dimensional matrix of pixel co or dinates and lifetime histogram channels causes serious problems Even with modern memory chips this ap proach is limited in image size and or number In addition it is expensive and implies loss of information about the individual photon arrival times To solve the problem much more elegantly the TTTR data stream generated by the PicoHarp 300 can contain markers for synchronization information derived from an imaging device e g a scan controller For this purpose the control ports of the PicoHarp 300 provide three TTL in puts for synchronization signals in addition to those for routing Note that the low cost model PicoHarp 300E does not have this feature The figure below illustrates how the external marker signals are recorded in the data stream event ool ooo oO O0 Lt ba start stop t r rr pe ct Le oa oo ae od time tag i amp amp amp amp het amp amp route R R R Rb R R H marker Mi Mla Bullets denote a photon blue pulses denote a marker signal The external markers are treated almost as if they were regular photon event records A special channel code allows to distinguish true photon records from the marker records Software reading the TTTR file can thereby filter out the markers e g for line and frame clock in imaging applications This makes possible to reconstruct the 2D image from the stream of TTTR records since the relevant XY positi
114. ng a plain T2 mode measurement except count rate and progress display However for the special case of Fluorescence Correlation Spectroscopy there is a real time correlator for T2 data Using custom software it is also possible to analyze incoming data on the fly Even on line correlation can be implemented Obviously this requires efficient processing and possible restrictions in average count rate The PicoHarp software installation CD contains demo programs to show how T2 mode files can be read by custom software The implementation of custom measurement programs requires the PicoHarp program ming library which is available as a separate option T3 Mode In T3 mode channel 0 is dedicated to a sync signal As far as the experimental setup is concerned this is similar to histogramming mode The main objective is to allow high sync rates up to 84 MHz which could not be handled in T2 mode Accommodating the high sync rates in T3 mode is achieved as follows First the sync divider is employed as in histogramming mode This reduces the sync rate so that the channel dead time is no longer a problem The remaining problem is now that even with a divider of 8 the sync rate is still too high for collecting all individual sync events like ordinary T2 mode events Considering that sync events are not of primary interest the solution is to record them only if they arrive in the context of a photon event on channel 1 This is the same as the original T
115. ng figure shows TCSPC histograms obtained with this setup Ex citation source was a PDL 800 B with a 440 nm laser head running at 10 MHz repetition rate D data Coum6b phd PicoHarp III x File Edit View Help Daja S S 2 l el els del wl gt lal d Coumarin bin Ethanol 440 ex 520 em 35 0 timens Input frps inputi fcps Histog ftps r Total Counta r bax Count at Time ms FuwHhd ms Route 1 00e 007 g 45e 004 g Aen Ebbe Hie A ga 16 530 2776 Sum READY F Page 11 For help press Fl PicoQuant GmbH PicoHarp 300 software v 2 3 The narrower blue curve represents the system IRF here dominated by the detector PMT The other curve red is the fluorescence decay from a solution of Coumarin 6 in ethanol a fluorescent dye with fairly short fluorescence lifetime 2 5 ns Note that the count rate was adjusted to lt 1 of the laser rate to prevent pile up The plot in a logarithmic scale shows the perfect exponential nature of the decay curve as one would expect it Note that this is ob tained even without a deconvolution of the IRF 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 e 1 e g 27 183 10 000 In this particular experiment this resulted in a lifetime estimate of 2 5 ns in perfect agree ment with published results for this dye For a precise measurement one would p
116. on of e g a scanner can be determined during the data analysis The data generated is nearly free of redundancy and can therefore be transfered in real time The image size is unlimited both in XY and in count depth Since there are up to three such sychronization signals all imaging applications can be implemented and even other experiment control signals can be recorded This marker scheme is a very special feature of the PicoQuant TCSPC electronics To date the PicoQuant Time Harp and PicoHarp are the only TCSPC systems that supports this powerful measurement mode It may be worth noting that this technology enabled PicoQuant GmbH to develop the leading edge MicroTime 200 Fluorescence Lifetime Microscope Section 8 3 2 contains the detailed pin assignments of the PicoHarp s router control ports two sub D con nectors labeled RT and CTRL Here we focus only on the signals of relevance for TTTR mode and mark ers Three TTL compatible inputs accept the synchronization signals that will be recorded as markers The active edges of these signals can chosen in the software settings dialog available through the Toolbar Both high and low state must be at least 100 ns long The period may therefore in principle be as short as 200 ns but data bus throughput constraints will apply Each marker creates an additional TTTR record so that one must ensure not to swamp the data stream with too many marker records Markers also take precedence over photon
117. one timing circuit on a first come first serve principle This works fine because the probability of receiving a photon in one sync period must be kept on the order of 1 or 2 anyway which is a prerequisite for avoiding pile up in TCSPC If two photons arrive during the same sync period only the first one will be accepted The router recognizes in which channel it received a photon and delivers that information to the PicoHarp via a dedicated router control port The next photon can be on any other channel whichever comes first Because of the inherent count rate limitations in TCSPC this meth od of multiplexing can in fact increase the overall counting efficiency This is because pile up effects do not occur across channels and the overall count rate can thereby be larger than the permitted 1 to 2 per channel Even with mutually inflicted dead time losses the counting efficiency of each channel remains close to 1 and the efficiency over all channels is substantially larger than that of a single channel system The only limitation multiplexing routing implies is that the total count rate the time digitizer can handle must be shared by the four channels Because of the single photon conditions there are consequently low probabilities of photons impinging on two or more detectors simultaneously The router uses a time window of some ns during which a decision is made as to whether a count is valid This is the case only if there was a signal from a
118. onstantly improve and update the software for our instruments This includes updates of the con figurable hardware FPGA Such updates are important as they may affect reliability and interoperability with other products The software updates are free of charge unless major new functionality is added It is strongly recommen ded that you register for email notification on software updates If you wish to receive such notification please email your name and the serial number of your PicoQuant product s to info picoquant com You will then receive update information with links for download of any new software release Bug Reports and Support The PicoHarp 300 system has gone through extensive testing Nevertheless it is a very cutting edge product and we would like to offer you our support in any case of problems with the system Do not hesitate to con tact PicoQuant in case of difficulties with your PicoHarp Should you observe errors or bugs caused by the PicoHarp system please try to find a reproducible error situation E mail a detailed description of the problem and all relevant circumstances especially other hard ware installed in your PC to info picoquant com Please run msinfo32 to obtain a listing of your PC config uration and attach the summary file to your error report Your feedback will help us to improve the product and documentation When you submit an error report referring to measurement data you have taken attach the origi
119. osition The count enable signal is effective in all modes It may be used to synchronize the acquisition with external con ditions If external disabling is not desired the pin must be held high or left open This signal is used by the router to identify valid counts It can not be used by external applications if a router is connected Pins 3 is a TTL output that indicates that the ctc is active measurement running Pins 4 5 and 9 accept the TTL compatible synchronization signals that will be recorded as markers in TTTR mode The pins are internally pulled down so that they are inactive when left unconnected The active edge is chosen in the software settings dialog Rise fall times must be 10 ns or faster Both high and low state must be at least 100 ns long The clock period may therefore in principle be as short as 200 ns but data bus throughput constraints will apply Each marker creates an additional TTTR record so that one must en sure not to swamp the data stream with too many marker records Markers also take precedence over photon records so that excess marker traffic can suppress photon records In fast imaging applications it is therefore recommended not to use a pixel clock but a line clock only Because each photon has a time tag it Page 68 PicoQuant GmbH PicoHarp 300 software v 2 3 is usually not necessary to use an additional pixel clock Note that the same signals can also be fed in via the CTRL connector so that the
120. pin numbers LEZEA J TELEF H 100600 Ob RT PicoHarp 300 rev 2 0 router and control connectors viewed from the outside of the chassis RT Connector The 15 pin male sub D connector labeled RT is the connector for the PHR 402 403 routers See section 5 3 on Routed Measurements for details on routing and router connection It also carries marker signals for TTTR mode Therefore if you wish to use a PHR 40x together with marker input a dedicated Y cable for the simultaneous connection to the RT port can be used available from PicoQuant Note that this cable must not be extended Only required marker signals may be extended to connect the marker source In principle these signals can also be fed in via the CTRL connector but a special DVI plug will be needed The following table shows the RT connector s pin assignments Pin UO Description TTL in count enable 1 3 TTLout ctc active 4 TTL in marker 1 input TT TR mode only 5 TTL in marker 2 input TTTR mode only 9 TTL in marker 0 input TTTR mode only 11 out 5V 0 5V lt 200 mA 12 GND ground 14 GND ground Pin 1 accepts a TTL signal that is used by the router to mark counts as valid It can also be used to extern ally enable disable recording of incoming counts Counting is enabled when pin 1 is held high This mechan ism can be used to blank out periods where e g an external scanner is moving to the next p
121. r pins are used for communication with dedicated accessories PHR 40x routers etc and must not be connected otherwise Appropriate cables are available from PicoQuant CTRL Connector While the RT connector is primarily for the connection of routers the larger female 26 pin connector labeled CTRL is for generic control signals such as markers in TTTR mode The following table shows its pin as signments Pin UO Description 1 9 10 GND ground 2 8 out 5V 0 5V lt 200 mA Se out 3 3V 0 5V lt 200 mA 12 TTL in marker 1 input TT TR mode only 11 TTL in marker 2 input T TTR mode only 17 TTL in marker 0 input T TTR mode only 18 TTL in count enable 26 TTLout ctc active Pins 1 9 and 10 are the common ground for the TTL signals and the DC supply lines They are also connec ted to the chassis Pins 2 and 8 provide a 5 V DC supply voltage external electronics can use Under no circumstances must this lines be shorted or in total loaded in excess of the specified maximum current Note that the maximum current on the 5 V line must be shared with any loads connected at the 5 V line of the RT connector Pins 3 and 7 provide a 3 3 V DC supply voltage external electronics can use Under no circumstances must this lines be shorted or in total loaded in excess of the specified maximum current Pin 18 accepts a TTL signal that is used by the router to mark counts as valid It can al
122. ramming in on board memory Other modes are available as upgrade options For instance two different Time Tagged Time Resolved TTTR modes allow to record each photon event separately with the individual arrival times to provide unlimited flexibility in off line data analys is such as burst detection and time gated or lifetime weighted Fluorescence Correlation Spectroscopy FCS as well as picosecond coincidence correlation The PicoHarp 300 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 PicoHarp software The latter supports automated measurement of Time Re solved Emission Spectra TRES The PicoHarp hardware is offered in two versions The standard product PicoHarp 300 provides more fea tures The PicoHarp 300E is a leaner variant targeting cost sensitive applications where some functionality is not required The features that distinguish the PicoHarp 300 from the 300E model are support for routing devices for simultaneous measurements with several detectors and interfacing with external hardware via TTL marker signals The PicoHarp software runs on all recent 32 bit Windows PC platforms from Windows 2000 to XP and Vista as well as on 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
123. red 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 For the PicoHarp corresponding firmware upgrades must be purchased to make use of these features Details are described in section 5 4 on Time Tagged Mode Measurements Forward and Reverse Start Stop Mode For simplicity it is most convenient to assume that the time delay measurement is directly causal Le the laser pulse causes a photon event and one measures the time delay between laser pulse and the sub sequent photon event However most conventional TCSPC systems need to give up this logical concept be cause 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 3 to 2 us any excitation rate in excess of 0 5 to 3 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
124. rformed If for any reason the initializ ation becomes invalid e g if the monochromator was turned manually and subsequently accidentally reaches the end point switch the nitialization page will be activated and access to the Manual and TRES pages will be blocked Manual Monochromator Control The Manual page of the dialog can be used to position the monochromator manually at a given wavelength The desired position can be entered in the Position edit box Clicking Apply or Pressing lt Enter gt will cause the monochromator to move to the specified position Alternatively the spin buttons right to the edit box can be used to modify the position The step width of this action is defined in the A delta edit box Pressing ar row buttons will make the change take effect immediately entering values by keyboard require pressing Ap ply or lt Enter gt to show an effect J Mono ST 9030 E d Manual TRES Initialization Position nm Pe UD 7 DC Stepping UU nm Page 37 PicoQuant GmbH PicoHarp 300 software v 2 3 If there is a large difference between the current monochromator position and the position entered the movement will take some time If you wish to interrupt this process press the Stop button The monochro mator immediately will slow down and stop The deceleration is necessary to preserve the calibration Running TRES Measurements The TRES page of the dialog contains the controls used to set up an automa
125. rp 300 labelled Channel 0 and Channel 1 In case of time resolved fluorescence experiments with a pulsed ex citation source the sync signal must be connected to Channel 0 and the detector signal must be connected to Channel 1 If coincidence correlation experiments between two detector signals are to be carried out con nect one detector each to the two inputs Both inputs are dynamically terminated with 50 Q internally Note that they need negative going input signals Use quality 50 QO coax leads with appropriate connectors For in terfacing 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 sig nal sources 50 Q in your experimental setup The PicoHarp 300 inputs accept negative pulses going from zero to max 1 V Both inputs should be operated with similar pulse amplitudes to minimize crosstalk The optimum range is 100 to 500 mV Most PMT and MCP detectors will require a pre amplifier to reach this level TTL SPAD detectors Perkin Elmer SPCM AQR must be connected through a pulse inverter Pi coQuant 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
126. rs on that computer even if they have limited access rights The PicoHarp 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 PicoHarp software in mul tiple sessions at the same time The PicoHarp software 2 3 is also suitable for x64 versions of Windows XP and Vista where a suitable 64 bit device driver will be installed The PicoHarp software as such runs as a 32 bit application on WOW64 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 perform ance of this software and associated documentation License and Copyright Notice With this product you have purchased a license to use the PicoHarp 300 software on a single PC 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 disas semble the software or part thereof You may use and modify demo code to create your own software Ori ginal or mod
127. s int32 1 or 4 NumberOfBoards int32 reserved now 1 ActiveCurve int32 curve no used for last measurement MeasurementMode int32 O interactive 1 reserved 2 T2 3 T3 SubMode int32 O oscilloscope 1 integration 2 TRES RangeNo int32 O base resol 1 x2 2 x4 3 x8 4 x16 x128 Offset int32 offset in ns AcquisitionTime int32 acquisition time in ms StopAt int32 stop at this many counts StopOnOvfl int32 stop if overflow occurred 1 otherwise 0 Restart int32 restart 1 otherwise 0 DisplayLinLog int32 lin 0 log 1 Display TimeAxisFrom int32 lower time axis bound of display in ns Display TimeAxis T 0 int32 upper time axis bound of display in ns DisplayCountAxisFrom int32 lower count axis bound of display DisplayCountAxisTo int32 upper count axis bound of display The following block will appear in the file for each i 1 to 8 DisplayCurve i MapTo int32 number of memory block to map this curve to DisplayCurve i Show int32 O don t show 1 show end of block The following block will appear in the file for each i 1 to 3 Param i Start float used for automated measurements e g TRES Param i Step float reserved for automated measurements Param i End float reserved for automated measurements end of block RepeatMode int32 reserved for automated measurements RepeatsPerCurve int32 reserved for automated measurements RepeatTime int32 reserved for automa
128. should be a narrow peak as opposed to the flat distribution of uncorrelated light This re quires an experimental setup that delivers the signals at the two input channels with a more or less constant relative delay For the PicoHarp in histogramming mode this delay must be such that the Channel 1 detect or signal comes later than the Channel 0 sync signal forward start stop mode In addition the delay must be chosen so that it fits in the measurement range of the histogrammer max 33 us To obtain such a timing it may be necessary to make adjustments by inserting cable delays of appropriate lengths in either channel dependent on the delays in detector laser sync or any other signal paths electrical as well as optical If you have an appropriate setup installed and a measurement running you can slightly vary all levels in cluding sync for best timing response If there is no clear optimum for any level return to the center of its stable working range For optimization purposes you also may want to switch to a higher resolution If the rough setup is complete and a time correlated signal peak is present in the histogram the Full Width Half Maximum FWHM of the signal peak is displayed on line as a figure of merit You can also adjust your axis limits to optimize the display Note that you can double click all the rate meters to enlarge them as separate windows This is useful for optical alignment work if the PC screen is in some distance
129. single detector In the case of multiple simultaneous signals the events will be discarded This mechanism ensures a cross talk between channels of typically lt 0 01 at 500 000 counts sec In order to keep the crosstalk low it is recom mended not to exceed 500 000 counts sec per channel Because of the multiplexing nature of the routing mechanism and the TCSPC dead time it is generally not possible to perform short distance timing between individual routing channels e g for coincidence correla tion experiments However cross correlations on the time tags 100 ns resolution of individual channels can be performed very elegantly from a single data file This is of great value in Fluorescence Correlation Spec troscopy FCS Using a router does not normally change the time resolution or the IRF width although at high count rates some degradation may be observed Also due to dead time the count rates recorded on individual channels will drop as the count rates on other channels increase to very high numbers Therefore routing must be used with extra care in regard to single photon statistics This is usually not an issue at kHz count rates e g in single molecule spectroscopy In a routed setup the detectors are each directly connected to one router input via 50 Q SMA cables no at tenuator The router output is directly connected to the PicoHarp input channel 1 no attenuator The routers must also be connected to the PicoHarp 300 via a
130. sition times In this case the histogram continues to grow over a longer time and the display is updated at regular intervals so that the accumulation process can be observed To start a measurement with the current control panel settings use the start button GO on the toolbar or press lt ALT gt G To stop a measurement use the stop button on the toolbar or press lt ALT gt S Note that a measurement may automatically stop and or restart dependent on the current settings of stop on overflow and restart in the control panel Allow a warming up period of 20 to 30 minutes dependent on lab temperature before using the PicoHarp for final measurements You can use this time for set up and preliminary measurements Click the calibrate button after warming up Acquisition Rezal ps Offset ns E Tre Block Time coe Tre Block m o gt wr The rate meters bottom of main window permit visual control of the data acquisition They are often too small to view from further away e g when adjusting the optical setup You can in this case simply double click the rate meter of interest This opens a large meter display that you can then size and move on the screen as you like Once you have established standard settings for your experimental setup you may want to save them to a file The control panel settings can then be recalled at any time The program stores all settings together with the histogram data of those
131. so be used to extern ally enable disable recording of incoming counts Counting is enabled when pin 1 is held high This mechan ism can be used to blank out periods where e g an external scanner is moving to the next position The count enable signal is effective in all modes It may be used to synchronize the acquisition with external con ditions If external disabling is not desired the pin must be held high or left open This pin can not be used if a router is connected Pins 11 12 and 17 accept the TTL compatible synchronization signals that will be recorded as markers in TTTR mode The pins are internally pulled down so that they are inactive when left unconnected The active edge is chosen in the software settings dialog Rise fall times must be 10 ns or faster Both high and low state must be at least 100 ns long The clock period may therefore in principle be as short as 200 ns but data bus throughput constraints will apply Each marker creates an additional TT TR record so that one must ensure not to swamp the data stream with too many marker records Markers also take precedence over photon records so that excess marker traffic can suppress photon records In fast imaging applications it is therefore recommended not to use a pixel clock but a line clock only Because each photon has a time tag it is usually not necessary to use an additional pixel clock Pin 26 is a TTL output that signals when the ctc is active i e when a measurement
132. ss They can be found in the subfolder Filedemo_ in your chosen installation directory Page 58 PicoQuant GmbH PicoHarp 300 software v 2 3 8 2 2 T2 Mode File Format PicoHarp data files from T2 Mode pt2 are binary files which contain both the setup parameters and the actual event data The setup data is in the file header which is in the same format as in standard interactive mode files This is to allow common reading routines for the header Note that this leads to some header val ues that are not meaningful in T2 Mode In order to safely recognize a T2 Mode file apart from the file extension a program reading in these files must read and interpret the value MeasurementMode which is 2 in T2 Mode Format version 2 0 pt2 files are structured as shown in the table below Note that the format version is not necessarily the same as that of the creating software version Data Item Type Description Ident char 16 the string PicoHarp 300 FormatVersion char 6 currently 2 0 since Sept 2006 CreatorName char 18 for this software PicoHarp Software CreatorVersion char 12 currently 2 3 0 0 April 2009 FileTime char 18 the file creation date and time CR LF char 2 Ox0D 0x0A carriage return line feed Comment char 256 any ASCII string NumberOfCurves int32 not meaningful in T2 Mode BitsPerRecord int32 32 RoutingChannel
133. st Typical fluor escence 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 sig nal 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 20 ps time steps This is hard to achieve with ordinary electronic transient recorders of reasonable dynamic range Secondly the light avail able 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 one 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 cycles one can reconstruct the single cycle decay profile from single photon events collected over many cycles The method is based on the repeti
134. stem will NOT work through USB 1 0 All current PCs should provide USB 2 0 connectivity Make sure the drivers for the USB 2 0 host controller are correctly in Stalled If this is not ensured USB 2 0 interfaces may only run as USB 1 0 interfaces Older versions of Win dows may not have the appropriate drivers so you may need to install them from the mainboard driver disk For older PCs without USB 2 0 support it is possible to install USB 2 0 adaptor cards However such com puters may often be outdated in many other respects so if possible consider replacing them See sec tion 8 3 on Hardware Technical Data for requirements recommendations Always use a USB cable rated for high speed USB 2 0 The cable length allowed by USB 2 0 must not exceed 5 metres 16 ft For best reli ability we recommended to use the provided cable of 3 metres length Note that the USB specification does not allow cable extensions other than dedicated active extension cables or HUBs The PicoHarp device should work flawlessly through USB 2 0 high speed HUBs This is also a valid way of extending the maxim um cable length beyond 5 metres After a HUB another cable of up to 5 metres is allowed Note however that chains of multiple 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 two SMA connectors located on the front panel of the PicoHa
135. sync divider gt 1 WARNING_INPO RATE_TOO_ HIGH You have selected T2 mode and the pulse rate at input channel O is higher than 5 MHz The measurement will d inevitably lead to a FiFo overrun There are rare measurement scenarios where this condition is expected and the warning can be disabled Examples are very short measurements where the FiFo can absorb all data WARNING_INP1_RATE_ZERO No counts are detected at input channel 1 In histogramming and T3 mode this is the photon event channel and the measurement will yield nothing without this signal You may d d sporadically see this warning if your detector has a very low dark count rate In that case you may want to disable this warning Page 54 PicoQuant GmbH PicoHarp 300 software v 2 3 Warning Histo Mode T2 Mode T3 Mode WARNING_INP1_RATE_TOO_HIGH If you have selected T2 mode then this warning means the pulse rate at input channel 1 is higher than 5 MHz The measurement will inevitably lead to a FiFo overrun There are rare measurement scenarios where this condition is expected and the warning can be disabled Examples are very short measurements where the FiFo can absorb all data d d d In histogramming and T3 mode this warning is issued when the input rate is gt 10 MHz This will probably lead to deadtime artefacts There are rare measurement scenarios where this condition is expected and the warning can be disabled WARNING_INP_RATE_RATIO Th
136. t 1 count rate as displayed by rate meter Crv i HistCountRate int32 histogramming count rate as displayed by rate meter Crv i IntegralCount int64 total count as displayed by meter Crv i Reserved int32 reserved Crv i DataOffset int32 file offset of corresponding histogram data Crv i RouterModelCode int32 O none 1 PHR_402 2 PHR_403 3 PHR_800 Crv i RouterEnabled int32 1 enabled O disabled routing off Page 5 PicoQuant GmbH PicoHarp 300 software v 2 3 Crv i RtCh_InputType int32 O custom 1 NIM 2 TTL Crv i RtCh_InputLevel int32 in mV Crv i RtCh_InputEdge int32 1 rising O falling Crv i RtCh_CFDPresent int32 O not prsent 1 present Crv i RtCh_CFDLevel int32 in mV Crv i RtCh_CFDZeroCross int32 in mV end of block The following block will appear in the file for each i 1 to lt NumberOfCurves gt j 1 to lt Channels gt as indicated in the curve header Counts i j uint32 the actual histogram data leftmost channel first end of block These items are stored as they were set displayed last They may not relate to the data stored These items may vary and thereby change the file structure dynamically A program must interpret these before reading in further data See section 8 2 4 for the data types used Also please note that the software setup will install a ready to use set of examples in C Pascal Delphi Lazarus and Matlab for PicoHarp phd_ file acce
137. t TTL compatible synchronization signals that will be recorded as markers in TTTR mode The pins are internally pulled down so that they are inactive when left unconnected The active edge is chosen in the software settings dialog Rise fall times must be 10 ns or faster Both high and low state must be at least 100 ns long The clock period may therefore in principle be as short as 200 ns but data bus throughput constraints will apply Each marker creates an additional TT TR record so that one must ensure not to swamp the data stream with too many marker records Markers also take precedence over photon records so that excess marker traffic can suppress photon records In fast imaging applications it is therefore recommended not to use a pixel clock but a line clock only Because each photon has a time tag it is usually not necessary to use an additional pixel clock All other pins are used for communication with dedicated accessories or future extensions and must not be connected otherwise All information given in this manual is reliable to our best knowledge However no responsibility is assumed for possible inaccuracies or omissions Specifications and external appearance are subject to change without notice PicoQuant GmbH Unternehmen fur optoelektronische Forschung und Entwicklung Rudower Chaussee 29 IGZ 12489 Berlin Germany Telephone 49 0 30 6392 6560 Fax 49 0 30 6392 6561 PicoQuanr email info picoquant com eae
138. t preview toolbar offers you the following options Print Bring up the print dialog box to start a print job Next Page Preview the next printed page Prev Page Preview the previous printed page One Page Two Page Preview one or two printed pages at a time Zoom In Take a closer look at the printed page Zoom Out Take a larger look at the printed page Page Setup Change the layout of the printed page Close Return from print preview to the editing window Page 51 PicoQuant GmbH PicoHarp 300 software v 2 3 7 Problems Tips amp Tricks PC Performance Issues The PicoHarp device and its software interface are a complex real time measurement system demanding appropriate performance both from the host PC and the operating system This is why a fairly modern CPU and sufficient memory are required A modern graphics interface is also recommended Screen resolution should be at least 800x600 At least a 1 GHz processor 512 MB of memory and a fast hard disk running in DMA mode are recommended In order to maintain correct interaction between the measurement hardware and the display of histogram curves and user input the operating system s message passing mechanism is used It is recommended not to overload the system by running other processes in the background while measuring with the PicoHarp The PC s own occasional network activity should be no problem but running the machine e g as a server for other PCs is not recommended I
139. tart gt Run gt msinfo32 In the System Informa tion utility inspect Software environment gt System Drivers to check if the PicoHarp device driver PQUSB SYS PQUSB64 SYS on x64 is correctly installed and running You can also repeat the driver and software installation if necessary Make sure the software is not installed in multiple places If problems persist disconnect the device uninstall the software and repeat the setup pro cedure If this does not resolve the problem see section 7 on Problems Tips amp Tricks for support 3 5 Uninstalling the Software Before uninstalling the PicoHarp software you should back up all PicoHarp data files you created in the PicoHarp directory Do not manually delete any program files from the installation folder as it will render a clean uninstall im possible To uninstall the PicoHarp software from your PC you may need administrator rights dep on Windows ver sion and security settings Choose Start gt Settings gt Controlpanel gt Software and select PicoQuant Pico Harp300 vx x for uninstallation This will remove all files that were installed by the PicoHarp setup program but not the user data that may have been stored If there was user data in the program folders or any sub folders 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 unin Sta
140. tc is available via the lt F1 gt key Help Topics command This command opens a browser offering a tree view like list of contents and a help page Browse through the contents with your mouse or the cursor keys lt Enter gt opens closes chapters lt CurUp gt lt CurDown gt turns the pages The help page to the right of the browser will always be held up to date Shortcuts Keys lt gt if online help was last recently opened in topics mode lt Alt gt H T Help Index command This command opens a browser offering a index list of keywords and a help page Browse through the con tents with your mouse or the cursor keys lt Enter gt opens the current item lt CurUp gt lt CurDown gt navig ates through the index Shortcuts Keys lt F1 gt if online help was last recently opened in index mode lt Alt gt H I Help Search command This command opens a browser offering a means of a full text search on words or word groups over the whole online help volume You can enter more specific search pattern using the operators AND OR NEAR and NOT Shortcuts Keys lt ET gt if online help was last recently opened in search mode lt Alt gt H S Activate Context Help Changes the mouse cursor mode to context help mode You can choose context sensitive help for a visual element in the GUI by simply clicking on it Shortcuts Keys Soh fic gt ae AVC PHC About PicoHarp command Use this com
141. ted measurements RepeatWaitTime int32 reserved for automated measurements ScriptName char 20 reserved for automated measurements The following block will appear in the file for each i 1 to lt NumberOfBoards gt currently this must be 1 Brd i Hardwareldent char 16 currently PicoHarp 300 Brd i HardwareVersion char 8 currently 1 0 or 2 0 Brd i HardwareSerial int32 hardware serial number Page 59 PicoQuant GmbH PicoHarp 300 software v 2 3 Brd i SyncDivider int32 input channel 0 Sync input divider Brd i CFDZeroCross0 int32 input channel 0 CFD zero cross level in mV Brd i CFDLevel0 int32 input channel 0 CFD discriminator level in mV Brd i CFDZeroCross1 int32 input channel 1 CFD zero cross level in mV Brd i CFDLevel1 int32 input channel 1 CFD discriminator level in mV Brd i Resolution float resolution in ns Brd i RouterModelCode int32 O none 1 PHR_402 2 PHR_403 3 PHR_800 Brd i RouterEnabled int32 1 enabled O disabled routing off The following block will appear in the file for each j 1 to lt NumberOfRouterChannels gt currently this must be 4 Brd i RtCh j InputT ype int32 O custom 1 NIM 2 TTL Brd i RtCh j nputLevel int32 in mV Brd i RtCh j InputEdge int32 1 rising O falling Brd i RtCh j CFDPresent int32 O not present 1 present Brd i RtCh j CFDLevel int32 in mV Brd i RtCh j CFDZCross int32 in mV
142. ted scanning of the monochro mator The start and stop wavelengths of the scan can be entered in the corresponding edit boxes the A delta box defines the step width of the scan The scan starts always exactly at the Start value If the Stop position cannot be reached by an integer multiple of the step width the scan stops at the last position within the start stop range If the start position is larger than the stop position the scanning direction will be re versed The maximum range and number of wavelength steps is determined by the physical characteristics of the monochromator as well as the maximum number of memory blocks to record histograms in 512 Again pressing the spin buttons will apply the change in the corresponding edit boxes immediately entering values manually require pressing lt Enter gt or the Apply button JE Mono ST 9030 x Manual TRES Initialization Start nm E 460 00 Ss Sto GN 660 00 ET W IRF at CX Stepping 520 00 mm The IRF can be measured automatically as part of the scan at a separately chosen wavelength To do so check the box entitled IRF at and enter the wavelength at which to measure the IRF in the corresponding edit box The monochromator will go to the IRF position at the beginning of the TRES measurement After the IRF was measured the actual TRES scan will be performed The IRF will always be recorded in memory block 0 while the wavelength scan always starts in block 1 Note that
143. the sync input from the laser and Channel 1 for the detector Here we focus on the latter more common case Perform the following steps to set up the input discriminators Setting Up the Sync Input Input Channel 0 For typical fluorescence decay measurements the PicoHarp needs an electrical sync signal from the light source The PDL 800 family of diode lasers from PicoQuant provide this signal directly 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 sync signal must consist of pulses with falling active edge e g a NIM type signal is appropri ate This is a steep negative pulse 0 5 to 10 ns wide of typically 800 mV into 50 Q The PicoHarp can ac tually handle 1 V but amplitudes in excess of 800 mV may cause excess interference and crosstalk between the input channels Amplitudes around 200 to 800 mV are best in terms of timing accuracy and lowest histogram ripple Another rule of thumb for lowest signal distortion is to use signals of similar amp litude on both channels SMA inline attenuators of suitable bandwidth can be used to adjust this The ad justable CFD level is restricted to 800 to 0 mV Initially you should set it to half of the sync pulse amplitude The zero cross setting should be set to 10 mV initially Unless you are sure what kind of signal your sync source delivers use a fast oscilloscope 50 Q input to check the pulse shape
144. ting the offset gt 0 Page 43 PicoQuant GmbH PicoHarp 300 software v 2 3 Use this set of input controls to specify the desired offset Units are Nanoseconds ns Possible offsets are in the range from 0 to 100000 ns Type the value as an integer in the edit box and press lt Enter gt The off set value can be entered in steps of one nanosecond Alternatively use the spin control next to the edit box to increment decrement the current value In this case changes take effect immediately without pressing lt P a cer Internally the offset is subtracted from each start stop measurement before it is used to address the histo gram channel to be incremented Therefore increasing the offset means shifting the signal on the screen to the left towards earlier times Note that the offset has no effect on the relative timing of laser pulses and photon events It merely shifts the region of interest where data is to be collected The relative timing of laser pulses and photon events can only be controlled by means of cable delays e g in order to shift your fluorescence decay within the sync frame so that it is not truncated Acquisition Time edit box and spin control Set the desired measurement time here Units are seconds s The permitted range is 0 001 to 360000 in steps of 0 001 Type the desired value in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment decrement the current value In t
145. tion of that version File formats in future software releases are subject to change without notification 8 2 1 Interactive Mode File Format The standard PicoHarp data files from interactive mode phd are binary files which contain both the setup parameters and the actual histogram data In order to safely recognize an interactive mode file apart from the file extension a program reading in these files must read and interpret the value Measurement Mode which is 0 in interactive mode Format version 2 0 phd files are structured as shown in the table below Note that the format version is not necessarily the same as that of the creating software version Data Item Type Description Ident char 16 the string PicoHarp 300 FormatVersion char 6 currently 2 0 since Sept 2006 CreatorName char 18 for this software PicoHarp Software CreatorVersion char 12 currently 2 3 0 0 April 2009 FileTime char 18 the file creation date and time CR LF char 2 Ox0D 0x0A carriage return line feed Comment char 256 any ASCII string NumberOfCurves int32 number of curves histograms stored in this file BitsPerHistogBin int32 normally 32 RoutingChannels int32 1 or 4 NumberOfBoards int32 reserved now 1 ActiveCurve int32 curve no used for last measurement MeasurementMode int32 O interactive 1 reserved 2 T2 3 T3 SubMode int32 O oscilloscope
146. tive precisely timed registration of single photons of e g a fluorescence signal The reference for the timing is the corresponding excitation pulse As a single photon sensitive de tector a Photo Multiplier Tube PMT or a Single Photon Avalanche Diode SPAD can be used 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 record ing 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 at all in many cycles The diagrams below illustrate how the histogram is formed over multiple cycles A a e ms A A Fo w laser pulsef NM a many cycles do not A gt 1 produce a photon I fluorescence photon i fluorescence photon P F TCSPC start stop time start stop times histogram no photon registered no photon registered E WR first photon no photon registered time second photon result after many cycles Page 5 PicoQuant GmbH PicoHarp 300 software v 2 3 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 referred to as time channels In practice the registration of one photo
147. tive mode to a standard PicoHarp data file phd Reloading T3 mode files serves as a quick diagnostic tool only For T2 mode files such a feature is not avail able 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 SymPho Time Further specialized analysis can be performed by dedicated custom software If you wish to save the cost for the commercial TT TR 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 PicoHarp installation disks and will be in stalled by the software setup Also see section 8 1 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 addi tion to extracting the bit fields the software must step through the whole file and interpret the overflow re
148. to switch the high voltage supply of PMTs off and allow their electrodes to dis charge Their high voltage charge may damage the pre amplifier Observe the allowed input signal levels in cluding those of the pre amplifiers Again in a new experimental setup to be absolutely sure please check your detector pulses as well as the pre amplifier output with a fast oscilloscope Do not over illuminate the detector to avoid damage Start timing is on the leading edge so it should be steep Ringing and overshoot should be as small as possible All signals should have rise times of no more than 2 ns Slower signals will degrade timing accuracy Very slow may not be seen by the PicoHarp at all If the signals are appropriate connect the detector Preliminary adjustments can actually be done with un correlated light e g daylight For safety of your detector use strongly attenuated light even when the de tector is off In the beginning do not use a router to keep things simple Start the PicoHarp software and open the Control Panel In the Control Panel section for Channel 1 initially set the CFD Discriminator Level to 30 mV and the CFD Zero Cross level to 10 mV The rate meter next to the sync rate meter will now show the channel 1 input rate Make sure that there is actually a signal coming in to the Channel 1 input some strongly attenuated light on the detector and try to adjust the CFD Discriminator Level To monitor this watch the count rate meter
149. traviolet 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 re lated 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 Micro Channel Plate PMT MCP A Micro Channel Plate PMT consists of an array of glass capillaries 5 to 25 um 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 10 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 usuall
150. ttings affect only the real time correlator The raw T2 or T3 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 al low 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 correlation results you should save them here If you need to perform further or more thorough FCS ana lysis on the collected data you can use the SymphoTime software from PicoQuant Page 34 PicoQuant GmbH PicoHarp 300 software v 2 3 On the right hand side there are controls for the axis ranges of the correlator display Note that they affect only the display Collected data is always complete independent from the axis settings In contrast the se lection box subsmpl has an effect on the correlator results It determines how many tau points are calcu lated The correlator works in a logarithmic multiple tau scheme and subsmpl specifies the number of linear subsamples in each log stage A higher number of subsamples increases the resolution of the
151. uld occur In case of a FIFO overrun the measurement must be aborted be cause data integrity cannot be maintained However on a modern and well configured PC a sustained aver age count rate of 5 Mcps is possible This total transfer rate is fully available for input channel 1 For all prac tically relevant fluorescence detection applications this is more than sufficient Page 30 PicoQuant GmbH PicoHarp 300 software v 2 3 For maximum throughput T3 mode data streams are normally written directly to disk There is no immediate data visualization during a plain T3 mode measurement except count rate and progress display However for the special case of Fluorescence Correlation Spectroscopy there is a real time correlator for T3 data Using custom software it is of course possible to implement other algorithms for the analysis of incoming data on the fly Obviously any real time analysis requires efficient processing and possible restrictions in average count rate The PicoHarp software installation CD contains demo programs showing how T3 mode files can be read by custom software The implementation of custom measurement programs requires the PicoHarp programming library which is available as a separate option Running a plain TTTR Mode Measurement A TTTR mode measurement T2 or T3 mode will typically be started after all control panel settings have been tested in normal interactive histogramming mode oscilloscope or integration Th
152. urned into the ROM of the PicoHarp An upgrade is possible at any time It always includes both T2 and T3 mode System Requirements In cases where the Time Tagging modes are to be used with high continuous count rates gt 500 kcps the PC system must meet some special performance criteria that usually are insignificant in standard interactive use of the PicoHarp The reason for this is the relatively large amount of data being generated in TTTR mode In order to prevent an overflow in the recording this data must be transferred to the hard disk in real time This requires a modern PC with a fast I O subsystem A CPU running at least at 1 GHz is recommen ded For sufficient performance in TTTR mode a modern IDE or SATA hard disk with 7200 rom and 8 MB cache is a good and economical choice typically 160 GB and more Also suitable are modern SCSI hard disks with PCle or on board controller Another option for maximum throughput are RAID systems which however must be carefully chosen and configured At the same time the USB subsystem must be con figured correctly In order to deliver maximum throughput the PicoHarp 300 uses state of the art USB 2 0 bulk transfers This is why the PicoHarp must rely on having a working USB 2 0 host interface USB 2 0 host controllers of modern PCs are usually integrated on the mainboard For older PCS they may be upgraded as PCI cards Throughput is usually limited by the host controller and operating system not th
153. uted operation the block number shown in the control panel E Hz indicates the first block of four belonging to one measurement This is why in routed operation you can only change the block number in steps of four In order to see the data being collected in all four histograms you need to make sure the active block in the control panel and the next three blocks are mapped to display curves and switched on show in the trace mapping dialog The rate meters at the bottom of the main PicoHarp window also have a slightly different meaning when routing is enabled Only the input rate meter is as usual it shows the Route count rate at the PicoHarp s channel 1 input which is common for all routing channels Sum For the other meters there is a selector switch on the very right of the rate meters It l allows to assign the rate meters to either the sum of all routing channels or to one single routing channel click the up down arrows to make the selection When you open the Trace Mapping Dialog you will notice that after a routed measurement four curves have been filled with data and their details view button has become active The details shown in this dialog correspond to the information stored in the curve header of a phd file If a router was used in the measurement the router model PHR xxx will be shown in the details panel If you take measurements with the router connected but routing disabled the counts from all det
154. ving a working USB 2 0 host interface USB 2 0 host controllers of modern PCs are usually integrated on the mainboard For older PCs they may be upgraded as PCI cards Throughput is usually limited by the host controller and operating system not the PicoHarp Make sure that the host controller drivers are correctly installed otherwise they may be running only in USB 1 0 mode This may require using the mainboard manufacturer s driver disk rather than relying on Windows drivers alone Do not run other bandwidth demanding devices on the same USB interface when working with the PicoHarp USB cables must be rated for USB 2 0 high speed Older cables do not meed this requirement and can lead to errors and malfunction Similarly some PCs have poor internal USB cabling so that USB sockets at the front of the PC are sometimes unreliable Histogram Artefacts Disturbing histogram ripple is strongly dependent on the quality of the input signals Try to deliver the best possible signal quality with clean and reasonably steep active edges and without overshoot and ringing It is recommended to use detector and sync signals of similar amplitude to avoid excess crosstalk If the PDL 800 B picosecond diode laser is used attenuating the sync pulses by 10 dB immediately at the laser driver may reduce histogram ripple set CFD level accordingly Always use good quality 50 Q components and coax leads with proper shielding and correct termination We recommend cables of t
155. w lower limit BB je le 7 AB Iw upper limit Accoumul tef update subsmpl WW 8 C Dac Ce Int Reset Acquisition Settings f T2 Mode Elapsed Time ChO Cnt Rate Chi CrtRate Records Ce T3 Mode 00 01 00 4 00e 007 2 7e 005 13244467 fo T Wam overnite ready to start e Append numbers c stmp sdefault pt3 ce START etap CANCEL je eg les Te4 jee Tez Je Save Status The dialog section Cor Ax Bx allows to select individual routing channels for the correlation A and B are the virtual input channels to be correlated Provided that a router is installed all routing channels that are ticked in columns A resp B will be used in the corresponding correlation channel If no router is installed only Rt 1 is meaningful If your selection is incomplete 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 nt If the latter is chosen one can manually reset the correlator by clicking the Reset button Note that all these se
156. ware Settings Dialog This setting does not equally apply in oscil loscope mode If you see no counts make sure you are not running oscilloscope mode with acquisition times longer than a few seconds Once Channel 1 count rates gt 0 are being displayed you should then also see counts appearing in the histogramming rate meter provided your measurement range is large enough Make sure the offset is set to 0 If there are histogramming counts but no histogram is building up on the screen check the time and count axis bounds It is best to start with a wide display range and then narrow it down Similarly a logarithmic count axis setting is the safest way to start because you see even small histograms If your FWHM figures are wider than expected make sure the histograms are not overflowing peak count 65535 A shorter acquisition time and or the setting stop on overflow helps to avoid this Do not set the stop at level to less than 65536 unless you have experimental reason to limit the count height In the process of set up you should observe the warning icon that may appear at the bottom right of the main window When the PicoHarp software is running with functional hardware it continuously collects information about the input signals and the current acquisition settings If these settings to gether with the input rates indicate possible errors the software will activate the warning icon The warning icon can be clicked to display a list
157. warning Note that the time axis labelling is precise in terms of relative information only At offset 0 the absolute time difference between the inputs is close to the shown times but may be off by some tens of picoseconds Off sets gt 0 change the placement of the acquisition window and the true time differences are then off accord ingly The time axis settings will determine the number of channels exported via clipboard copy paste Only the channels within the displayed time range will be used Time Axis Maximum edit box and spin control Here the end of the displayed time axis can be set Units are nanoseconds ns Type the value as an in teger in the edit box and press lt Enter gt Alternatively use the spin control next to it to increment or decre ment the current value In this case changes take effect immediately without pressing lt Enter gt The maxim um allowed here is 33555 ns If the entered value becomes smaller than the current time axis minimum an error message appears When using the spin control violating the current time axis minimum is automatic ally prevented Instead of decrementing the minimum value further the system beeps a warning Observe the notes on time axis interpretation given in the paragraph TimeAxis Minimum above The time axis settings will determine the number of channels exported via clipboard copy paste Only the channels within the displayed time range will be used Count Axis Minimum edit
158. when an offset gt 0 is set even though the sync period 1 Rate0 can d d be covered by the measurement time span without using an offset The offset may lead to events getting discarded There are some measurement scenarios where this condition is intentional and the warning can be disabled If any of the warnings you receive indicate wrong pulse rates the cause may be inappropriate input settings wrong pulse polarities poor pulse shapes or bad connections If in doubt check all signals with a scope Note that all count rate dependent warnings relate to the PicoHarp s native input channels If you are using a router remember that all routing channels share the PicoHarp s input channel 1 and hence create a cumulated common rate at that channel Page 55 PicoQuant GmbH PicoHarp 300 software v 2 3 8 2 Data File Formats While for many purposes the ASCII export function via the clipboard is sufficient and easy you also may want to access the PicoHarp data files via custom programs This section provides the file structure specific ations for your reference Note that the file structures of PicoHarp 300 files are different from TimeHarp NanoHarp files Programs that need to process the files should check the string dent in the file header In order to safely recognize a PicoHarp file this string should be checked in any case It is also necessary to check the version strings and read the data according to the format specifica
159. y Note that it is also only an approxima tion obtained by multiplying the G 0 approximation with the average count rate on both virtual correlation channels A and B Dependent on the chosen routing channels in A and B this may lead to figures that are not truly molecular brightnesses however as an adjustment aid they should be useful in any case The real time correlation is very demanding in terms of CPU load The correlator is implemented in a way that will make use of multiple processor cores It is therefore beneficial to use one of the recent dual or quad core CPUs All other aspects of TTTR data collection with correlator preview are the same as in plain TTTR mode Top ics such as using external markers or how to use the data files should be looked up there Page 35 PicoQuant GmbH PicoHarp 300 software v 2 3 5 6 Time Resolved Emission Spectra In time resolved fluorescence research it is often of great interest to observe time dependent spectral shifts and decay changes in the context of solvent relaxation dynamics and general spectral evolution This re quires recording Time Resolved Emission Spectra TRES ideally with automated wavelength scan and TC SPC data collection under full software control In order to measure emission spectra in combination with fluorescence lifetime the PicoHarp software provides an automated TRES measurement mode This mode allows to control a monochromator via a step per motor and automated
160. y can be used even if a router is connected Pins 11 provides a 5 V DC supply voltage external electronics can use Under no circumstances must this line be shorted or loaded in excess of the specified maximum current Note that the maximum current must be shared with any loads connected at the 5 V line of the CTRL connector Pins 12 and 14 are the common ground for the TTL signals and the DC supply line They are also connected to the chassis All other pins are used for communication with dedicated accessories PHR 40x routers etc and must not be connected otherwise Appropriate cables are available from PicoQuant CTRL Connector While the RT connector is primarily for the connection of legacy routers the larger DVI I connector labeled CTRL is for connecting the PHR 800 router It also carries generic control signals such as markers in TTTR mode The following table shows its pin assignments Since most of them require low voltage differential sig nals LVDS it is recommended not to use this port for custom control signals Pin UO Description 1 2 LVDSin route 3 GND ground 8 TTLout ctc active 9 10 LVDSin route 2 11 GND ground 14 out 5V 0 5V lt 200 mA 15 GND ground 16 TTL in router detection 17 18 LVDSin route 1 19 GND ground 22 GND ground 23 24 LVDSin count enable C1 TTL in marker bit O input TT TR mode only C2 TTL in marker bit 1 input
161. y outperforming PMTs Good but also expensive MCPs can achieve timing uncertainties as low as 25 ps Microchannel plates are in this re spect the best match for the PicoHarp 300 but they are limited in permitted count rate and provide lower sensitivity towards the red end of the spectrum compared to suitably optimized APDs Avalanche Photo Diode APD APDs are semiconductor devices usually restricted to operation in the visible to infrared part of the spec trum Generally APDs may be used for ultra low light detection optical powers lt 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 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 and difficult to fo cus 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 Perkin Elmer SPCM AQR provide a timing accuracy of 400 ps selected and are specified to have a quantum effi
162. ype RG223 with double braided and silver plated screen Check your setup for ground loops Grounding different system components PC detector detector power supply diode laser driver etc at different points can induce con siderable noise in the ground lines Because signal return paths may share the same ground lines this noise is copied into the signal lines and causes increased timing jitter and or histogram ripple Network cables and mobile phones may also be sources of surprising noise Use properly impedance matched power splitters reflection free T pads if signals must be fed to multiple 50 Q inputs Never use ordinary Tees All accessor ies are available from PicoQuant PMT detectors should be connected through a suitable high speed preamplifier available from PicoQuant MCP PMT detectors should be connected through an amplifier with slightly higher gain e g PAM 102 M from PicoQuant TTL SPAD detectors Perkin Elmer SPCM AQR must be connected through a pulse inverter PicoQuant SIA 400 Page 52 PicoQuant GmbH PicoHarp 300 software v 2 3 A problem often overlooked is pile up and dead time related histogram distortion It becomes noticeable if the detector count rate exceeds 2 of the sync rate This is why high excitation rates are so important Pile up is an inherent problem of TCSPC and not a fault of the hardware The only way to handle it safely is to maintain count rates lt 1 to 2 of the sync rate Pile up may to som

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