Cornelius | DPC 230 | Specifications | Cornelius DPC 230 Specifications

High Performance
Photon Counting
User Handbook
DPC-230
16 Channel
Photon Correlator
Becker & Hickl GmbH
(c) Becker & Hickl GmbH
Becker & Hickl GmbH
April 2008
High Performance
Photon Counting
Tel.
+49 / 30 / 787 56 32
FAX
+49 / 30 / 787 57 34
http://www.becker-hickl.de
email: info@becker-hickl.de
DPC-230
16 Channel Photon Correlator
Photon correlation down to the ps range
16 fully parallel time-resolved photon-counting channels
16 LVTTL inputs for SPADs or 4 CFD inputs for PMTs
Recording of absolute photon times
Autocorrelation within 16 LVTTL or 4 CFD channels
Cross-correlation between any pairs of LVTTL or CFD channels
Full correlation down to the picosecond range
FCS and anti-bunching from one experiment
3-channel TCSPC mode with 165 ps time channel width
Multiscaler operation of 15 LVTTL or 3 CFD channels
Imaging capability in TCSPC and multiscaler modes
Time-tag data available in all modes
Available as single PCI cards or laptop-based stand-alone systems
II
Becker & Hickl GmbH
Nahmitzer Damm 30
12277 Berlin
Germany
Tel. +49 / 30 / 787 56 32
FAX +49 / 30 / 787 57 34
http://www.becker-hickl.com
email: info@becker-hickl.com
April 2008
This handbook is subject to copyright. However, reproduction of small portions of the material in scientific papers or other non-commercial publications is considered fair use under the
copyright law. It is requested that a complete citation be included in the publication. If you
require confirmation please feel free to contact Becker & Hickl.
Contents
Contents
Introduction ...............................................................................................................................................1
Principle of Data Acquisition ....................................................................................................................1
Time-to-Digital Conversion.................................................................................................................1
DPC-230 Architecture .........................................................................................................................2
Absolute Timing ............................................................................................................................2
Relative Timing..............................................................................................................................3
Operation Modes.......................................................................................................................................5
Absolute-Timing Modes ......................................................................................................................5
Intensity Traces ..............................................................................................................................5
Photon Counting Histograms .........................................................................................................5
Fluorescence Correlation ...............................................................................................................6
Relative Timing Modes .......................................................................................................................8
Multichannel Scaler Mode .............................................................................................................8
TCSPC Mode.................................................................................................................................9
TCSPC with Absolute Timing .......................................................................................................10
Imaging ..........................................................................................................................................11
Installation.................................................................................................................................................13
Computer .............................................................................................................................................13
Software Installation ............................................................................................................................13
Hardware Installation...........................................................................................................................14
Driver Installation................................................................................................................................14
Software Start ......................................................................................................................................15
Starting the SPCM Software without a DPC-230 Module...................................................................16
Operating the DPC-230 .............................................................................................................................17
LVTTL Inputs .....................................................................................................................................17
Connecting SPAD and PMT Modules to the LVTTL Inputs.........................................................17
Synchronisation to Light Sources via the LVTTL Inputs...............................................................18
Marker Pulses.................................................................................................................................18
CFD Inputs ..........................................................................................................................................18
Connecting PMTs to the CFD Inputs .............................................................................................19
SPADs............................................................................................................................................20
Synchronisation Pulses from Light Sources ...................................................................................20
Typical Applications .................................................................................................................................21
Fluorescence Decay Measurements .....................................................................................................21
Luminescence Decay Measurement in the Microsecond Range ..........................................................23
Fluorescence Correlation .....................................................................................................................24
Picosecond Fluorescence Correlation..................................................................................................28
Anti-Bunching .....................................................................................................................................29
Fluorescence Lifetime Imaging............................................................................................................31
Luminescence Lifetime Imaging in the Microsecond Range ...............................................................34
SPCM Software.........................................................................................................................................37
Configuring the SPCM Main Panel .....................................................................................................37
Changing Between Different Instrument Configurations ...............................................................38
Display and Trace Parameters........................................................................................................38
Resizing and Positioning the Display Windows.............................................................................39
Cursors in the Display Windows of the Main Panel.......................................................................40
Link to Data Analysis.....................................................................................................................40
Trace Statistics ...............................................................................................................................41
Status Information..........................................................................................................................41
System Parameters of the DPC-230.....................................................................................................43
Operation Mode .............................................................................................................................43
Specification of the Time-Tag Date File........................................................................................44
Name of Time-Tag Data File .........................................................................................................45
Configuring the Runtime Display...................................................................................................45
Configuring the Inputs ...................................................................................................................46
CFD and SYNC Parameters...........................................................................................................47
SYNC Frequency Divider ..............................................................................................................47
TDC Parameters.............................................................................................................................48
Saving Setup and Measurement Data ..................................................................................................49
III
IV
Loading Setup and Measurement Data ................................................................................................50
Predefined Setups ................................................................................................................................51
Converting FIFO Files .........................................................................................................................53
Format of Time-Tag Data Files ...........................................................................................................55
Specification..............................................................................................................................................59
References .................................................................................................................................................61
Index..........................................................................................................................................................63
Introduction
The DPC-230 photon correlator card records absolute photon times in up to 16 parallel detection channels. Depending on how the photons are correlated, fluorescence correlation (FCS),
fluorescence cross correlation (FCCS), photon counting histograms, or waveforms of the light
signals are obtained. In combination with optical scanning time-resolved images can be recorded. The maximum time resolution of the recording is 165 ps per time channel.
The DPC-230 is available as a single PCI card for installation in a standard PC, or as a compact ‘Simple-Tau DPC’ system based on a laptop computer with an extension box, see Fig. 1.
Fig. 1: DPC-230 card (left) and Simple-Tau DPC system (right)
The DPC-230 especially targets on a new type of fluorescence correlation spectroscopy that
records correlation over a time interval of 10 orders of magnitude [12]. Thus, singlet decay
rates, rotational relaxation rates, rate constants of conformational changes, triplet transition
and decay rates, and diffusion times of free fluorophores and fluorophores bound to proteins,
lipids or nanoparticles can be obtained within a single measurement.
Moreover, the DPC-230 can be used for recording luminescence decay curves or luminescence lifetime images from the sub-nanosecond to the millisecond range, for LIDAR experiments, FCS, time-of flight mass spectroscopy, or any other experiments based on acquiring
the temporal distribution of detection events in a large number of detector channels.
The DPC-230 is operated by the same software as the bh SPC modules [2]. Although the system parameters and operation modes of the DPC differ from those of the SPC modules the
general software philosophy is the same. Moreover, the DPC can be used with the same detectors, detector assemblies, detector controllers, excitation sources, and optical systems as the
SPC modules. This handbook should therefore considered an addendum to the ‘bh TCSPC
Handbook’ available on www.becker-hickl.com [2].
Principle of Data Acquisition
Time-to-Digital Conversion
The recording process in the DPC-230 is based on a digital TDC (‘Time to Digital Converter’)
principle. A TDC uses the delay of fast CMOS gates as a timing reference. A reference pulse
is cycling within a ring structure consisting of a number of similar CMOS gates, see Fig. 2.
When a photon (or any other input event) is detected the position of the reference pulse in the
2
Principle of Data Acquisition
ring is read, and used to determine the detection time. Times longer than the reference cycle
time are determined by counting the reference cycles.
G1
Ring Oscillator
G2
G3
Gn
Counter
Readout
Register
Input
Pulse
D
C
D
C
D
C
D
C
D
C
Encoder
fine time
coarse time
Fig. 2: Principle of a digital TDC. The time of the input pulse is derived from the location of a reference pulse
cycling within a ring oscillator
Compared with the TAC-ADC principle used in fast TCSPC devices [1, 2] a TDC delivers
coarser time-channels. A TDC can, however, be built at a lower price and with considerably
lower power consumption. More important, a large number of TDC channels can be synchronised to obtain comparable photon times in a large number of recording channels.
DPC-230 Architecture
Absolute Timing
A general block diagram of the DPC-230 is shown in Fig. 3. The device has two TDC chips,
each of which contains eight TDC channels. Both TDC chips are synchronised via a common
clock oscillator. Both TDC chips can be operated either with 8 LVTTL (Low-Voltage TTL)
inputs or with two ECL (Emitter-Coupled Logic) inputs. The LVTTL inputs are compatible
with all commonly used single-photon avalanche photodiode (SPAD) detectors. The ECL
inputs are driven by constant-fraction discriminators (CFDs). The CFD inputs are compatible
with the output pulses of photomultiplier tubes (PMTs).
TDC Chip 1
In 1
PMT
CFD
TDC Channel1
In 1
In 2
In 2
LVTTL
PMT
LVTTL
CFD
TDC Channel2
In 3
LVTTL
TDC Channel3
In 4
.
.
.
.
In 8
LVTTL
.
.
.
.
LVTTL
TDC Channel4
FIFO
.
.
Bus
TDC Channel8
Clock Generator
TDC Chip 2
In 9
PMT
In 9 LVTTL
In 10 PMT
In 10 LVTTL
FIFO
2 million events
CFD
TDC Channel9
CFD
TDC Channel10
In 11 LVTTL
TDC Channel11
In 12 LVTTL
.
.
.
.
.
.
.
.
In 16 LVTTL
TDC Channel12
FIFO
FIFO
2 million events
.
.
TDC Channel16
Fig. 3: General architecture of the DPC-230
Interface
Principle of Data Acquisition
3
When a measurement is started the TDCs in all active channels simultaneously start running.
Any event detected at one of the 16 LVTTL inputs or at one of the four CFD inputs triggers a
time readout from the TDC of the corresponding channel. The time is first buffered in an internal first-in-first-out (FIFO) memory of the TDC chip. The data from the internal FIFO are
then written into an external FIFO that buffers up to 4 million events. The output of the external FIFO is read by the computer, and the data are transferred into the main memory or to the
hard disc. The double-buffered structure avoids loss of photons by read-write collisions.
Moreover, the external FIFO is large enough to buffer the photon data during possible background operations of the computer.
In the configuration shown in Fig. 3 the DPC delivers the times of the photons from the start
of the measurement. Such data are often called ‘time -tagged photon data’. The interpretation
of the data is merely a matter of the software. Thus, intensity traces, photon counting histograms, and fluorescence correlation and cross-correlation curves can be obtained (see
‘Operation Modes’ page 5).
Relative Timing
Relative timing is required if the waveform of an optical signal is to be recorded. The times of
the photons have then to be measured with reference to the pulses of the excitation source.
Relative timing can be achieved by recording reference pulses from the excitation source in
one of the TDC channels. The times of the photons in the excitation period are then obtained
by determining the differences of the photon times to the previous excitation pulse. This kind
of relative timing is used in the multichannel-scaler mode of the DPC (see ‘Multichannel Scaler Mode’, page 8).
Excitation pulse rates in the MHz range, as they are typical of TCSPC measurements, do,
however, cause a problem if direct relative timing is used: The high repetition rate of the reference pulses would saturate the TDC channel that is used for the reference. Therefore, the
DPC has a TCSPC configuration implemented that records only reference pulses of excitation
periods that contain valid photons. The DPC-230 architecture in the TCSPC mode is shown in
Fig. 4.
TDC Chip 1
In 1
CFD
TDC Channel1
In 2
CFD
TDC Channel2
FIFO
FIFO
2 million events
Bus
Clock Generator
TDC Chip 2
In 9
In 10
Reference
CFD
CFD
TDC Channel9
FD
REF
SYNC
TDC Channel10
FIFO
FIFO
2 million events
Fig. 4: DPC-230 architecture in the TCSPC mode
Interface
to
Computer
4
Principle of Data Acquisition
Three of the CFD channels are used for the detector signals. The CFD output pulses of these
channels are fed directly into the ECL inputs of the TDC chip. The fourth CFD input is used
for the reference from the laser. Before the reference pulses enter the TDC chip they pass a
frequency divider, FD, and a synchronisation circuit, SYNC. The divider ratio of FD is selectable and can be set to 1:1, 1:2, or 1:4. The subsequent synchronisation circuit transmits one and only one - synchronisation pulse if one of the first three CFDs has received a photon pulse
within the previous pulse period of the divided reference signal. Thus, TDC2 receives one
reference pulse when at least one photon was recorded in the previous 1, 2, or 4 excitation
periods. As a result, the reference pulse rate is reduced to less than the photon rate. The reduction is achieved without sacrificing any excitation periods or photons.
Pulse diagrams for FD = 1:1 and FD = 2:1 are shown in Fig. 5 and Fig. 6, respectively. With
FD = 1:1 the time of a photon is measured with respect to the next reference pulse. Thus, the
measurement time interval extends over one period of the light source.
With FD = 2:1 (Fig. 6) the photon time is measured with respect to the next or second next
excitation pulse, depending on whether the photon was detected in an even or uneven excitation period. Thus, the measurement time interval extends over 2 laser periods. A frequency
divider ratio of four results in a recording over four signal periods.
Reference,
from laser
Interval
recorded
Photons
Recorded
reference
pulse
Time of photon
Time of photon
Fig. 5: Reference synchronisation for FD=1:1. After the detection of a photon the next reference pulse is recorded. The time of the photon is measured with respect to the selected pulse.
Reference,
from laser
Reference,
after FD=2
Interval
recorded
Photons
Reference
after
SYNC
Time of photon
Time of photon
Fig. 6: Reference synchronisation for FD=2:1. The reference pulses are divided by a ratio of 2:1. After the detection of a photon the next pulse from the frequency divider is recorded. The time of the photon is measured with
respect to this pulse. The measurement time interval extends over 2 reference periods.
Operation Modes
Absolute-Timing Modes
In the ‘Absolute Time’ mode every photon is characterised by its time from the start of the
measurement and its input channel number. The interpretation of the data is merely a matter of
the software. The different ways of analysing the data and the presenting the results are described below. The results can be built up online from the incoming data stream, or off-line
from the time-tag data files. Please ‘Configuring the Runtime Display’, page 45.
Intensity Traces
The simplest way of interpreting the TDC data is to calculate intensity traces from the photon
times. The photon numbers within consecutive time intervals of the photon data stream are
determined, and displayed as functions of time, see Fig. 7. Although the procedure is trivial,
intensity traces can be used to determine on-off states of molecules, and to check whether an
experiment is running as expected.
Photons: Time-tag data
Intensity trace: Number of photons in consecutive time intervals
Fig. 7: Buildup of intensity traces from the time-tag data delivered by the TDCs
Photon Counting Histograms
The photon counting histogram (PCH) of an optical signal is obtained by recording the photons within consecutive sampling-time intervals and building up the distribution of the frequency of the measured counts versus the count numbers, see Fig. 8. For a classic light signal
of constant intensity the PCH is a Poisson distribution. If the light fluctuates, e.g. by fluctuations of the number of fluorescent molecules in the focus of a laser, the PCH is broader than
the Poisson distribution.
t
Number
of
t
intervals
containing
N photons
Number
of
photons
detected
time
PCH
Number of photons per
t
N
Fig. 8: Photon counting histogram. Left: Photons counted in successive sampling time intervals. Right: Histogram
of the number of time intervals containing N photons
A first characterisation of the PHC was given in 1990 by Qian and Elson [26]. The PCH delivers the average number of molecules in the focus and their molecular brightness. Several
molecules of different brightness can be distinguished by fitting a model containing the rela-
6
Operation Modes
tive brightness and the concentration ratio of the molecules to the measured PCH. The technique is also called ‘fluorescence intensity distribution analysis’, or FIDA. The theoretical
background is described in [9, 10, 15, 17, 21, 23, 24].
The PCH/FIDA technique can be extended for multi-dimensional histograms of the intensity
recorded by several detectors in different wavelength intervals or under different polarisation.
Multi-dimensional photon counting histograms have been shown to deliver a substantially
improved resolution of different fluorophores [16, 17]. With the large number of input channels available in the DPC-230 recording multi-dimensional PCHs is merely a matter of the
optical system.
Fluorescence Correlation
Fluorescence correlation spectroscopy (FCS) is based on exciting a small number of molecules in a femtoliter volume and correlating the fluctuations of the fluorescence intensity. The
fluctuations are caused by diffusion, rotation, intersystem crossing, conformational changes,
or other random effects. The technique dates back to a work of Magde, Elson and Webb published in 1972 [20]. Theory and applications of FCS are described in [8, 27, 28, 29, 30].
The (un-normalised) autocorrelation function G(τ) of an analog signal I(t) and the crosscorrelation function, G12(τ), of two signals I1(t) and I2(t) are
For photon counts, N, in consecutive, discrete time channels G(τ) and G12(τ) can be obtained
by calculating
G (τ ) = ∑ N (t ) ⋅ N (t + τ )
G12 (τ ) = ∑ N1 (t ) ⋅ N 2 (t + τ )
For a randomly fluctuating signal, I(t), the autocorrelation function G(τ) assumes high values
only if the values if intensity values, I, at a given time, t, and at a later time, t+τ are correlated.
Uncorrelated fluctuations of I cancel over the integration time interval. Similarly, G12(τ) assumes high values if the fluctuations in both signals, I1(t) and I2(t+τ), correlate with each
other. The drop of G(τ) and G12(τ) over the shift time, τ, shows how far the fluctuations are
correlated in time. The general behaviour of the auto- and cross-correlation functions is illustrated in Fig. 9.
I1(t)
I(t)
t
I2(t)
t
t
G( )
G12(
)
Fig. 9: General behaviour of the autocorrelation function, G(τ),of a signal I and the cross-correlation function,
G12(τ), of the signals I1 and I2
Absolute-Timing Modes
7
The autocorrelation (shown left) has a sharp peak at τ = 0. (The function correlates perfectly
with itself). The slow fluctuations in I(t) represent themselves in a high correlation at medium
τ. For τ longer than the typical time of the slow fluctuations G(τ) drops to very low values.
The cross-correlation function of the two signals, I1 and I2, is shown right. The slow fluctuations in I1 and I2 have the same general appearance. However, the noise in I1 and I2 is different. Therefore only the fluctuations correlate and give a noticeable contribution to G12(τ).
The equations shown above are appropriate to calculate correlation functions from analog
signals. The do, however, not well apply to time-tagged photon counting data recorded at high
time resolution. Such data do not display a continuous waveform as shown in Fig. 9 but are
rather a random sequence of individual detection events.
The general correlation procedure for time-tagged photon data is demonstrated in Fig. 10. In
typical TDC data, the time-channel width, T, is shorter than the dead time of the detector/photon counter combination. Therefore only one photon can be recorded in a particular
time channel. Consequently, N(t) and N(t + τ) can only be 0 or 1. The calculation of the autocorrelation function therefore becomes a simple shift, compare, and histogramming procedure.
The times of the individual photons are subsequently shifted by one time-channel interval, T,
and compared with the original detection times. The coincidences found between the shifted
and the unshifted data are transferred into a histogram of the number of coincidences, G, versus the shift time, τ. The obtained G(τ) is the (un-normalised) autocorrelation function.
Photon times
t
Number of coincidences
G
t
=T
=
2T
t
=
3T
t
Fig. 10: Calculation of the autocorrelation function from TCSPC time-tag data
The cross-correlation function between two signals is obtained by a similar procedure. However, the photon times of one detector channel are shifted versus the photon times of another
channel.
The result of the shift-and-compare procedure is not normalised. Normalisation can be interpreted as the ratio of the number of coincidences found in the recorded signal to the number of
coincidences expected for an uncorrelated signal of the same count rate. The normalised autocorrelation and cross-correlation functions are
Gn (τ ) = G (τ )
nT
N P2
with nT = total number of time intervals, Np = total number of photons, and
G12 n (τ ) = G12 (τ )
nt
N P1 N P 2
with nT = total number of time intervals, Np1 = total number of photons in signal 1, Np2 = total
number of photons in signal 2.
8
Operation Modes
The procedure illustrated in Fig. 10 yields G(τ) in equidistant τ channels. The width of the τ
channels is equal to the time-channel width of the DPC-230, T. The algorithm is known as the
‘Linear Tau’ algorithm. The equidistant τ channels result in an easily predictable statistics.
This is a benefit if a model function is to be fitted to the correlation function. Unfortunately,
for the small time-channel width of the DPC the algorithm would result in an extremely large
number of τ channels in G(τ), and in intolerably long calculation times.
The operating software of the DPC therefore applies binning steps to the photon data during
the correlation procedure. The general procedure is illustrated in Fig. 11.
photon data
t
Histogram of coincidences
shifted photon data
t+T
shifted photon data
t+2T
after
k
steps:
photon data
Binning 3:1
photon data
Histogram of coincidences
shifted photon data
n(t1)n(t2)
shifted photon data
Fig. 11: Correlation procedure with progressive binning
The procedure starts with a number of shift-and-compare operations as shown in Fig. 10.
However, after a number, kτ, of shift-and-compare operations the remaining photon data are
binned by a factor of three. (The factor of three is used to keep the new time intervals centred
at multiples of the original time channel width, T.) Then the shift-and-compare operation is
continued on the binned data. After every kτ operations a new binning step is performed. Thus,
the width of the τ channels increases progressively. The progression is the faster the smaller
the progression parameter, kτ is chosen. The procedure shown in Fig. 11 delivers similar results as the ‘multi-τ algorithm’ commonly used in conventional correlators [18].
Relative Timing Modes
In the relative timing modes reference pulses are recorded in one channel of the DPC-230. The
times of the reference pulses are recorded together with the photon times, and relative times
between the photons and the reference pulses are calculated.
Multichannel Scaler Mode
The multichannel-scaler mode records the waveform of a light signal, or, in the general case,
the density of the detected events as a function of the time within the period of a repetitive
excitation signal. In other words, the photon times are measured with reference to the pulses
Relative Timing Modes
9
of the excitation source, and the distribution of the events within the excitation period is built
up. Thus, the multichannel scaler mode is a relative-timing mode.
Multichannel scaler operation is illustrated in Fig. 12. The hardware structure is the same as in
the ‘absolute time’ mode. However, one channel of the DPC is used as a reference channel.
The reference channel receives synchronisation pulses from the light source. The other channels receive the single-photon pulses from the detectors. The data stream of the DPC contains
the times of the excitation pulses, and the times of the detected photons. From these data, the
operating software extracts the relative times of the photons since the last excitation pulse.
These times are used to build up the distribution of the photon density over this time. Because
several detectors can be active, several waveforms for the individual detectors can be obtained
within the same measurement.
Reference,
from laser
Reference,
from laser
Photons
Photons
t
t
Accumulation over many laser periods:
Pulse density versus time in signal period
t
Fig. 12: Multiscaler mode of the DPC. One channel records reference pulses from the excitation source, the other
channel records the photons. Relative times of the photons are determined, and curves of the photon density over
the time within the excitation period is built up.
The multichannel scaler mode is recommended for excitation rates up to about 1 MHz. For
higher excitation rates the high reference pulse rate can cause data transfer problems, and the
TCSPC mode should be used.
TCSPC Mode
The TCSPC mode was implemented for waveform measurements at high repetition rate. The
TCSPC mode measures the photon times with reference to the next excitation pulse. In classic
TCSPC the principle is known as ‘reversed start-stop’ technique [1, 2, 22]. The benefit of the
reversed start-stop principle is that reference pulses need to be recorded only for excitation
periods that contain valid photons. In classic TCSPC reversed start-stop avoids the problem of
excessive TAC start rates; in TDC-based instruments it avoids saturation of the TDC channel
of the reference. The configuration of the DPC-230 in the TCSPC mode is shown in Fig. 4,
page 3.
The build-up of the signal waveform in the TCSPC mode is illustrated in Fig. 13. In the typical TCSPC applications the count rate is considerably lower than the signal repetition rate.
Thus, there are a large number of signal periods in which no photons are detected. In these
periods the reference synchronisation circuit (see Fig. 4 and Fig. 5) suppresses the reference
pulses, and nothing is recorded. In signal periods that contain a photon both the photon and
the subsequent reference pulse are recorded. The software analyses the data stream, determines the relative times from the photons to the next reference pulse, and builds up the distribution of the photon density over the relative time. Except for a reversal of the time scale, the
10
Operation Modes
result is the waveform of the light signal. Because three input channels are available in the
TCSPC mode three signals from three detectors can be recorded simultaneously.
Reference
(not recorded)
Ref.
(not rec.)
Reference
(not recorded)
Photon
Time
Reference
(not recorded)
Reference
(not recorded)
Reference
(not recorded)
Photon Time
Reference
(not recorded)
Ref.
(not rec.)
Reference
(recorded)
Reference
(recorded)
Reference
(not recorded)
Photon
Time
Reference
(recorded)
Accumulation over many
excitation periods
Photon density vs. time in signal period
Photon time
Time in signal period
Fig. 13: Build-up of the signal waveform in the TCSPC mode
In contrast to TCSPC devices using the TAC/ADC principle the DPC is able to record several
photons per signal period. This ‘multi-hit’ capability of a TDC is often praised as a panacea
against pile-up distortions. However, in the typical fluorescence lifetime applications the lifetime is on the order of a few nanoseconds. The dead time of the detector/CFD/TDC combination is at least at the same order, if not longer (about 5 ns for PMTs and 50 ns for SPADs).
Thus, there is little chance for a single detector to record several photons within the fluorescence decay. In standard fluorescence applications with lifetimes on the order of a few ns the
multi-hit capability is therefore of little benefit. It is, however, important when fluorescence
decay functions in the 100 ns range or above are recorded, i.e. from quantum dots, semiconductors, or rare-earth chelates.
As all bh TCSPC devices, the DPC has a selectable frequency divider in the reference channel. The divider ratio determines the number of signal periods over which the signal is recorded (see Fig. 4 through Fig. 6). It should be noted here that a result containing several signal periods normally cannot be fully exploited by standard data analysis programs. Nevertheless, recording several signal periods can be useful when the time scale is to be checked by a
signal of known period, or when a TCSPC device is used as an optical oscilloscope [1, 2].
TCSPC with Absolute Timing
The time-tag data of the TCSPC mode still contain the absolute times of the detected photons.
Thus, a TCSPC measurement can be used to simultaneously obtain the waveform of the optical signal and the autocorrelation or cross-correlation within one or between different detector
channels [1, 2]. The waveforms of the detector signals are obtained as shown in Fig. 13, the
correlation curves as shown in Fig. 10. The result is the same as for the FIFO (or time-tag)
mode of the bh TCSPC mode where the fluorescence decay curves are obtained from the relative (‘micro’) time and the FCS curves from the absolute (‘macro’) times [2]. The results can
be built up online from the incoming data stream, or off-line from the time-tag data files.
Please see [2] and ‘Configuring the Runtime Display’ page 45.
Relative Timing Modes
11
Other applications of the absolute times in TCSPC data are multi-parameter single-molecule
spectroscopy [19, 25, 31], burst-integrated lifetime (BIFL) experiments, and fluorescence intensity and lifetime distribution analysis (FILDA) [23].
Imaging
The DPC-230 can be used for imaging applications. The sample is scanned by a focused laser
beam, the photons are detected with their absolute or relative times, and the image is built up
from the recorded data.
The imaging modes of the DPC-230 use the parallel architecture of the DPC module to record
both the photons and the synchronisation pulses from the scanner. Typically three scan synchronisation signals are used. A ‘Frame Clock’ edge indicates the start of a new frame, a ‘Line
Clock’ edge the start of a new line, and a ‘Pixel Clock’ the transition to a new pixel. The clock
edges are recorded in parallel with the photons, and with their absolute times. By identifying
the clock edges in the data stream every photon is assigned to the pixel the scanner was on in
the moment when the photon was detected. The method is similar to the FIFO Imaging mode
of multi-dimensional TCSPC, a technique routinely used for fluorescence lifetime imaging
(FLIM) with laser scanning microscopes [1, 2]. The principle is illustrated in Fig. 14.
Ti:Sa or ps diode
laser
Scan
head
Detector
Built up in computer memory
Light
Light
Photon times
Reference
from Laser
Frame Sync
Line Sync
Microscope
DPC-230
Pixel Clock
Photon Distribution
n (x, y, t)
Time in
decay curve
Laser Times
pixels
t
Y
Frame start times
Location
Line start times
Pixel start times
in
scan area
pixels
X
Fig. 14: DPC-230 Imaging: FLIM with laser scanning microscope
The laser scanning microscope scans the sample with a pulsed laser beam. The fluorescence
light is fed to a photon counting detector via a suitable optical port of the microscope. The
single-photon pulses of the detector are connected to one channel of the DPC-230. A second
channel records reference pulses from the laser. The relative photon times, i.e. the differences
of the photon times recorded in the photon and laser channel, are the times of the photons
within the fluorescence decay.
The frame clock, line clock, and pixel clock pulses from the scanner are connected to three
other channels of the DPC-230. By analysing the events recorded in these channels, the location of the laser beam in the scan area is obtained for each individual photon.
The fluorescence lifetime image is built up in the memory of the computer. A data array contains memory space for all pixels of the scan, and, within each pixel, memory space for a large
number of time channels within the fluorescence decay. The software extracts the relative
photon times and the locations from the data stream, and builds up a photon distribution over
these parameters.
Please note that the principle not only works for slow scanning with piezo stages but also for
fast scanning by galvanometer mirrors. In the typical fast-scan applications the pixel dwell
time is on the order of a few microseconds or less. The pixel rate is then higher than the pho-
12
Operation Modes
ton count rate. This makes the recording process more or less random. The recording is continued over as many frames as necessary to obtain the desired number of photons per pixel.
There are many modifications of the principle shown in Fig. 14. Imaging is possible both in
the multiscaler mode and the TCSPC mode. Other operations than FLIM calculation can be
applied to the photon and pixel data, and several detectors can be used simultaneously. By
storing the raw data, i.e. the photon times, the laser pulse times and the scan clock times, any
kind of processing can be applied to the data. The technique can therefore by used also for
multi-parameter fluorescence detection and correlation imaging [11, 19].
Installation
Computer
In principle, the DPC-230 module can be installed in a PCI slot of any Pentium PC. However,
the SPCM software runs the data transfer and the online-data processing in parallel ‘threads’.
To achieve a reasonable data transfer and processing rate the computer should be one with a
dual-core architecture.
The graphics card should have 1024 by 628 resolution or more. Both table-top and tower versions can be used. However, the computer must accept a full-size PCI card. It is recommended
to have two or three free PCI slots available. The DPC-230 can then be used in conjunction
with a bh DCC-100 detector controller card or a bh GVD-100 scan controller card.
The operating system can be Windows 2000, NT, XP, or Vista. The computer should have
1 Gigabyte of main memory. If the DPC is used for long measurements at high count rates a
main memory of 2 Gigabytes or more is recommended. The large memory helps to avoid hard
disc actions during the measurement and thus increases the maximum sustained count rate.
The DPC is operated via the SPCM software that is also used for the bh SPC modules. Although the SPCM operating software requires only a few Mb of hard disk space, much more
space should be available to save the measurement results. The time-tag data files can be as
large as several Gigabytes. It is therefore recommended to use a hard disc of several 100 Gb.
A DVD drive or another backup medium is also recommended.
Software Installation
The software installation starts automatically after the insertion of the installation CD. You
may also install or update the software form the web. In this case, open www.bekcerhickl.com, and click on the ‘Software’ button. On the ‘Software’ page (Fig. 15, left), click on
‘TCSPC Modules’, ‘Operating Software’. Then click on ‘Setup’ for SPC-830, SPC-730,
SPC-630, SPC-134, SPC-144, SPC-154 ((Fig. 15, right)). Download TCSPC_setup_web.exe
and start it.
Fig. 15: Software download from the web. Left: ‘Software’ page. Right: Page for downloading the SPCM software
14
Installation
Fig. 16: Installation panels
The installation works the same way as the installation from the CD. Check the boxes of the
software components you want to install (Fig. 16, right). The operating software of the DPC is
‘SPCM’. If you operate the DPC together with the DCC-100 detector controller you need also
‘DCC’. The SPCM and DCC software components are all you need for DPC standard applications. The components are free and can be installed without any password.
The installation package also contains a FLIM data analysis software, SPCImage, and DLL
libraries for the bh DPC, SPC and DCC modules. These components are not needed for DPC
standard applications. Their installation requires a licence number. You get this number if you
purchase these software components from bh. Please note that the DLL and Lab View libraries are only required if you plan to design your own experiment software. They are not required to run the SPCM software.
For details of the installation please see The bh TCSPC Handbook [2].
Hardware Installation
To install the DPC-230 module, switch off the computer and insert the board (and the
DCC-100 board, if you have one) into a free slot PCI slot. To avoid damage due to electrostatic discharge we recommend to touch a metallic part of the computer with one hand and
then to grasp the module at the metallic back shield with the other hand. This will drain any
potentially dangerous charge from you and the module. Then insert the module into a free slot
of the computer. Keep the DPC module as far as possible apart from loose cables or other
computer modules to avoid noise pick-up.
The DPC-230 modules has a PCI interface. Windows has a list of PCI hardware components.
On the start of the operating system, it automatically assigns the required hardware resources
to the components of this list. If you have several DPC or DCC modules in the computer each
module automatically gets its own address range.
Driver Installation
When the computer is started the first time with a DPC module Windows detects the new
module and attempts to update its list of hardware components. Therefore it may ask for
driver information from a disk. When this happens, put the installation CD into the drive and
select the drivers for Windows 2000, NT, XP an Vista. If you don’t have the installation disk
at hand you can also download the driver file from the bh web site, see Fig. 17.
Software Start
15
Fig. 17: Downloading the drivers from the bh web site
Open www.becker-hickl.com and click on ‘Software’. On the ‘Software’ page, click on
‘TCSPC Modules’, ‘Drivers’. Then click on ‘Setup’ and download the drivers.
Software Start
When the module is inserted and the driver is installed, start the SPCM Software. The initialisation panel shown in Fig. 18, left should appear. The installed modules (there can be up to
four) are marked as ‘In use’. The modules are shown with their serial number, PCI address
and slot number.
Important: If the DPC module is not found at this stage, e.g. because the driver was not installed, the software starts in an emulation mode (see below, ‘Starting the SPC software without an SPC module). What you see in this mode is generated by the software or loaded from a
file - it is not the data recorded by your DPC-230 module!
The software runs a simple hardware test when it initialises the module. If an error is found, a
message ‘Hardware Errors Found’ is given and the corresponding module is marked red. In
case of non-fatal hardware errors you can start the SPCM main window by selecting ‘Hardware Mode’ in the ‘Change Mode’ panel. Please note that this feature is intended for trouble
shooting and repair rather than for normal use.
When the initialisation window appears, click on ‘OK’ to open the main window of the SPCM
Software. At the first start the software comes up with default parameter settings which may
be not appropriate for your measurement problem. Therefore, changes may be required for
your particular application, please see ‘Configuring the SPCM Main Panel’, page 37.
Fig. 18: Starting the SPCM software. Initialisation panel (left) and main panel of SPCM (right)
When you exit the SPCM software after changing parameters, the system settings are saved in
a file ‘auto.set’. This file is automatically loaded at the next program start. So the system will
come up in the same state as it was left before.
16
Installation
Starting the SPCM Software without a DPC-230 Module
You can use the Multi SPC Software without a DPC module. In its start window the software
will display a warning that the module is not present, see Fig. 19, left.
Fig. 19: Startup panel in the simulation mode (without a TCSPC module)
To configure the software for the desired module type, click into the ‘Change Mode’ field and
select the module type (DPC-230) from the list which is opened (Fig. 19, right). Click on the
‘in use’ buttons for the modules you want to have active. Then click on ‘Apply’, ‘OK’. The
software will start in a special mode and emulate the DPC device memory in the computer
memory. You can set the system parameters, load, save, process, convert and display data, i.e.
do everything except for a real measurement.
Operating the DPC-230
LVTTL Inputs
The LVTTL inputs of the DPC-230 are designed to receive single-photon pulses from SPAD
(single-photon avalanche photodiode) modules. LVTTL means ‘low voltage TTL’, i.e. the
inputs are triggered by signals as low as +1.5 V. The pulse duration can be as short as 2 ns.
The location of the LVTTL inputs at the DPC board are shown in Fig. 20. The signals are fed
trough the back panel of the computer via cable adapters (Fig. 20, middle and right).
Fig. 20: Location of the LVTTL inputs at the DPC board (left), cable adapter (middle), DPC and adapters inserted in computer back panel (right)
Due to the limited size of the computer back panel MCX connectors had to be used for the
LVTTL inputs. Please contact bh if you need additional cables with MCX connectors.
The LVTTL inputs are overload-protected and accept input voltages up to +5V. The input
equivalent circuit is shown in Fig. 21.
100 Ohm
Input
to internal
circuitry
Termination
board
51 Ohm
Fig. 21: Equivalent circuit of the LVTTL inputs
The LVTTL inputs of the DPC-230 board are terminated with 50 Ω via a termination board.
The board is plugged into a connector close to the LVTTL inputs at the DPC board, see Fig.
20, left. We recommend to use the termination whenever the signal source is able to drive a
50 Ω load. For signals from standard TTL and LVTTL logics it may be necessary to remove
the termination. Operation without termination can cause reflections on the signal lines and
thus lead to double triggering. However, for standard TTL or LVTTL sources reflections are
damped by the source impedance and are thus rarely troublesome.
Connecting SPAD and PMT Modules to the LVTTL Inputs
The LVTTL inputs can be used for all commonly used SPAD modules and a number of PMT
modules with internal discriminators and TTL outputs. Some frequently used detectors are
listed in the table below.
18
Operating the DPC-230
Manufacturer Type
id Quantique
MPD
Perkin Elmer
Hamamatsu
id 100-xx
PDM-xxCT
SPCM-AQR
H7421-40
Pulse
Amplitude
+2 V
+2 V
+3.5 V
+3V
Pulse
Width
20 ns
20 ns
35 ns
30 ns
Remark
Versions of different area available
Use TTL output. Versions of different area available
GaAsP PMT module, QE = 40% at 500nm, large area
When using LVTTL- or TTL-compatible detectors, please make sure that the corresponding
DPC-230 input is configured for ‘rising edge’, see ‘Configuring the Inputs’ page 46.
Synchronisation to Light Sources via the LVTTL Inputs
When operated in the multichannel-scaler mode the DPC-230 needs a timing reference signal
from the light source. Pulses from commonly used PIN or avalanche photodiode modules are
usually not LVTTL-compatible. If the DPC has to be synchronised via a photodiode the circuit
shown in Fig. 22 can be used.
+5V
1k
Fast PIN
Photodiode
To DPC-230,
LVTTL Input
100n
1k
Fig. 22: Generating an LVTTL-compatible trigger signal via a PIN photodiode
Marker Pulses
Marker pulses can be used to identify external actions within the data stream delivered by the
DPC. Typical examples are pixel and line synchronisation pulses of a scanner. Marker pulses
can be connected to any LVTTL input that is not used for a detector. The pulses should be
LVTTL or TTL. Please note that only the low-high or the high-low transition of the signal
appears in the data. Thus, make sure that the DPC inputs are correctly configured, see
‘Configuring the Inputs’ page 46.
CFD Inputs
The DPC-230 has four CFD (constant fraction discriminator) inputs. These inputs are designed to trigger on single-photon pulses of PMTs. For best high-frequency behaviour, SMA
connectors are used. CFD The CFD inputs are shown in Fig. 23.
Fig. 23: CFD inputs
The pulse amplitudes at the CFD inputs should be negative and within a range of -50 mV to
-500 mV.
CFD Inputs
19
Connecting PMTs to the CFD Inputs
PMTs use secondary-electron emission to multiply a single photoelectron by a factor of 106 to
108. The output pulses of a PMT are negative, with a average amplitude between 10 mV and
100 mV and a duration between 300 ps and a few ns. Due to the random gain mechanism the
pulse amplitude varies randomly. The variation can be on the order of 1:10. Moreover, there
are a large number of small-amplitude background pulses from the dynode system of the
PMT.
A conventional discriminator would suppress the background pulses, but introduce a timing
jitter on the order of the duration of the leading edge of the pulses. Accurate triggering on
PMT pulses therefore requires a trigger circuit that not only suppresses pulses below a given
amplitude but also triggers at a time independent of the pulse amplitude. A CFD solves the
problem by a double-discriminator principle, see Fig. 24. One discriminator, D1, triggers
when the differentiated pulse crosses the signal baseline. The moment when the differentiated
pulse crosses the baseline is independent of the pulse amplitude. However, because the
threshold of the zero-cross discriminator must be close to the baseline, it also triggers to small
pulses and noise. Therefore a second discriminator, D2, is used to select input pulses above a
given amplitude. Only if the pulse amplitude is larger than a selectable threshold the pulse
edge of the zero cross discriminator is passed to the subsequent timing circuitry. Details of the
CFD design are described in [1, 2].
Input difference
voltage of D1:
Threshold
Delay
Input pulse
Zero Cross
+
- D1
Input
Delayed
pulse
D-FF
Enable
Zero cross
Threshold
+
- D2
Difference
Zero
cross level
Fig. 24: Constant-fraction discriminator (CFD)
The threshold of the DPC CFDs can be set from 0 to -500 mV. In principle, all commonly
used PMTs could be connected directly to the CFD inputs. There are, however, reasons why
this is not recommended:
PMTs can deliver pulses of extremely high amplitude. Such pulses can be caused by radioactive particles generating light pulses in the glass of the tube, by instability in the PMT, or by
illumination by strong laser pulses. Although the CFD inputs are overload-protected extremely strong pulses can damage the sensitive discriminator chips of the CFDs. Moreover,
when the PMT output is connected directly to the CFD there is no control about the output
current of the PMT. However, a PMT can easily be damaged by operation at excessive light
intensities. PMTs should therefore always operated via a preamplifier. The amplifier not only
protects the CFD against damage, it also delivers an overload warning if the maximum safe
output current of the PMT is exceeded. Please see [2, 3] for details.
The recommended operating conditions for a number of PMTs and PMT modules are shown
in the table below.
20
Operating the DPC-230
Manufacturer
bh
bh
Hamamatsu
Hamamatsu
Hamamatsu
Type
PMC-100-201)
PMC-100-011)
H7422P-401)
H5773P-00, -01
R3809U
Preamplifier
internal
internal
HFAC-26-2
HFAC-26-10
HFAC-26-01
HV / Gain
100 %2)
95%2)
90%2)
0.9V3)
-3000V
CFD Thresh.
-40 mV
-80 mV
-100 mV
-100 mV
-50 mV
Remark
cooled PMT module
cooled PMT module
cooled GaAsP PMT module
PMT module
MCP PMT
1) Operated via bh DCC-100
2) DCC-100 gain setting
3) Gain control voltage
Please note that the gain of a PMTs strongly depends on its operating voltage. Moreover, the
gain of different PMTs of the same type may be different by almost an order of magnitude.
The values given in the table above are typical values. To obtain best counting efficiency and
best IRF shape, please follow the instructions given in [2].
SPADs
Although the CFD inputs are primarily designed to accept single-photon pulses from PMTs
they can also be used for recording pulses from SPAD modules. Because the SPAD modules
have internal discriminators the threshold and zero-cross setting of the CFD have negligible
influence on the counting efficiency and the instrument response width. SPAD modules having an output for negative pulses can be connected directly to the CFD inputs; SPAD modules
with positive outputs need an A-PPI pulse inverter.
Manufacturer
id Quantique
MPD
Perkin Elmer
Type
id-100
PDM-xx-CD
SPCM-AQR
CFD Thresh.
-100 mV
-100 mV
-100 mV
CFD Zero cross
-20 mV
-20 mV
-20 mV
Remark
use A-PPI-D pulse inverter
use ‘Timing’ output
use 10 to 20dB attenuator plus A-PPI-D
Synchronisation Pulses from Light Sources
For TCSPC operation the timing reference signal has to be connected to CFD input number
four. The input accepts pulses of negative polarity. The amplitude should be 100 mV to
500 mV; the duration can be anywhere from 300 ps to 5 ns. Thus, synchronisation signals
from the bh BHL and BHLP diode lasers can be connected directly. For the bh BDL lasers an
A-PPI-D pulse inverter is required (see Fig. 25, left). Also synchronisation signals from Titanium-Sapphire lasers can usually be used. Depending on the polarity, an A-PPI-D pulse inverter may or not be required.
If synchronisation signals are not available from the light source, a suitable signal can usually
be generated by a bh PHD-400 photodiode module (see Fig. 25, right).
Fig. 25: Left: A-PPI-D pulse inverter. Right: PHD-400-N photodiode module
Typical Applications
Fluorescence Decay Measurements
A typical experiment setup for fluorescence decay measurement is shown in Fig. 26. The sample is excited by a bh BDL-SMC picosecond diode laser [5]. The pulse repetition rate is 20,
50, or 80 MHz, the pulse width between 50 and 90 ps. The laser is operated from a simple
+12V wall-mounted power supply. The laser power is controlled by a signal from the bh
DCC-100 detector controller [3].
Laser Switch Box
Synchronisation
A-PPI-D Pulse
Inverter
+12V
Delay
DPC-230 module
Trigger Out
bh BDL-SMC
ps Diode laser
Photon
SYNC
Detector
bh PMC-100
DCC-100 Detector Controller
Sample
Filter
PMC Power supply and
Overload Shutdown
Fig. 26: Fluorescence decay measurement
The fluorescence is separated from scattered excitation light by a bandpass filter. Please note
that the optical setup is shown simplified. In practice often a monochromator is used, and the
fluorescence light is transferred to the detection system by a lens. Moreover, a polariser may
be inserted in the beam path to remove the effect of rotational polarisation from the fluorescence decay. Please see [1, 2] for details.
The fluorescence is detected by a bh PMC-100 PMT module. The PMT module is controlled
by a bh DCC-100 detector controller. The DCC-100 delivers the power for the PMT and the
thermoelectric cooler of the PMC-100. It also provides overload shutdown of the detector if
the light intensity becomes too high [2, 3].
The photon pulses of the PMC-100 are fed into a CFD input of the DPC-230 module. More
PMC-100 modules can be added to the system and connected to the CFD inputs number 2 and
3. A synchronisation signal from the laser is connected to CFD input number 4. It is recommended that the synchronisation signal be delayed by a few meters of cable. With the right
delay, the synchronisation pulse arrives later than the pulse of a photon detected in the same
laser period. The location of the curves in the recorded time interval then becomes independent of the laser repetition rate [2].
For fluorescence decay measurement the DPC-230 is used in the TCSPC mode. It builds up a
photon distribution over the time in the laser pulse period. The typical DPC system parameters
are shown in Fig. 27. The measurement control parameters are located in the left part of the
system parameter panel. The operation mode is ‘TCSPC FIFO’, ‘Single’. The measurements
starts and stops by operator commands. If you want the measurement to stop after a specified
time, switch in ‘Stop T’, and specify the desired ‘Collection Time’.
22
Typical Applications
The right part of the panel shows the discriminator parameters of the CFD inputs and the TDC
parameters. The optimal discriminator settings depend on the detectors; the parameters shown
are typical of the bh PMC-100 module. The TDC parameters contain the time-channel width,
the number of time channels, and the time range of the recording. Moreover, the curves can be
shifted in time by applying an offset to the TDC times.
Fig. 27: System parameters for fluorescence decay measurement
The input configuration button opens the panel shown in Fig. 28. Both CFD channels of both
TDCs are activated. CFD 1, 2, and 3 are used as detector inputs. CFD number four is used as a
reference input for the laser pulses.
Fig. 28: Input configuration for TCSPC with up to three PMT detectors
The display of the curves is controlled by the trace parameters and display parameters, see Fig.
29. The trace parameters define which curves are to be displayed, and assign different colours
to the individual curves. The trace parameters shown in Fig. 29, left, display the data recorded
by a detector connected to channel 1, i.e. CFD 1. Curves of several measurements can be recorded into subsequent ‘pages’ of the data memory. The trace parameters shown display the
curves recorded into page 1 and page 2.
The scale of the display and other details of the display are defined in the display parameters,
see Fig. 29, right. A linear scale was selected, and the autoscale function was turned on. Two
decay curves recorded and displayed with the settings of Fig. 27 through Fig. 29 are shown in
Fig. 30.
Luminescence Decay Measurement in the Microsecond Range
23
Fig. 29: Trace parameters and display parameters recommended for fluorescence decay measurement
Fig. 30: Decay curves recorded with the setup parameters shown in Fig. 27 through Fig. 29
Luminescence Decay Measurement in the Microsecond Range
A suitable setup for luminescence decay measurements in the µs range is shown in Fig. 31.
For extremely long decay times the laser pulse repetition rate must be reduced to a few 10 kHz
or less. Using ps pulses at a repetition rate this low would result in an extremely low average
excitation power. It is therefore better to use excitation pulses with a duration of a few 100 ns
to several µs. The bh BDL-SMC diode laser is therefore operated in the CW mode [5]. The
laser is switched on and off periodically via its on/off input. To generate pulses of appropriate
duration suitable on/off signal is generated by a bh DDG-200 pulse generator module. The
pulse width and repetition rate of the excitation pulses is defined by the DDG-200 setup parameters.
Laser Switch Box
A-PPI-D Pulse
Inverter
CW
+12V
on/off
DPC-230 module
Power
Trigger Out
bh BDL-SMC
ps Diode laser
DDG-200 Pulse Generator
Module
Delay
A-PPI-D
pulse
inverter
Photon
SYNC
DCC-100 Detector Controller
Sample
Detector
bh PMC-100
Filter
or other pulse generator
PMC Power supply and
Overload Shutdown
Fig. 31: Luminescence decay measurement in the microsecond range
24
Typical Applications
The luminescence light is detected the same way as for fluorescence decay measurement. The
reference pulses for the DPC-230 are obtained from a second output of the DDG-200 pulse
generator.
For microsecond lifetime measurement the DPC-230 is operated in the multiscaler mode. The
system parameters are shown in Fig. 32. The other parameters are the same as for fluorescence
decay measurement. A typical result is shown in Fig. 33.
Fig. 32: System parameters for microsecond-luminescence decay measurement
Fig. 33: Luminescence decay recorded with the system parameters shown in Fig. 32. Left: Logarithmic scale.
Right: Linear scale
Fluorescence Correlation
Fluorescence correlation spectroscopy (FCS) is based on exciting a small number of molecules in a femtoliter volume and correlating the fluctuations of the fluorescence intensity. The
fluctuations are caused by diffusion, rotation, intersystem crossing, conformational changes,
or other random effects. The required femtoliter volume can be obtained one-photon excitation and confocal detection or by two-photon excitation. The technique dates back to a work
of Magde, Elson and Webb published in 1972 [20]. Theory and applications of FCS are described in [8, 27, 28, 29, 30].
Fluorescence Correlation
25
A typical optical setup is shown in Fig. 34. A CW laser beam is focused into the sample
through a microscope objective lens. The fluorescence light from the sample is collected by
the same lens, separated from the laser by a dichroic mirror, and fed through a pinhole in the
upper image plane of the microscope lens. Light from above or below the focal plane is not
focused into the pinhole and therefore gets substantially suppressed. With a high-aperture objective lens the effective sample volume is of the order of a femtoliter, with a depth of about
1.5 µm and a width of about 400 nm. A similarly small sample volume can be obtained by
two-photon excitation. Typical FCS instruments use several detectors in different wavelength
intervals or under different polarisation. Due to the similarities in the optical system, FCS is
possible with most confocal laser scanning microscopes [2].
CW
Laser
Detectors
Dichroic
Dichroic Pinhole
Dichroic
Polarising
beamsplitter
Detectors
Microscope
Lens
Fig. 34: Optical setup for FCS measurements
For good FCS results high detection efficiency is essential. The reason is the unusual signalto-noise behaviour of correlation experiments. In a fluorescence lifetime or FLIM measurement a 50% loss in efficiency can be compensated by increasing the acquisition time by a factor of two. In an FCS measurement a 50% loss in detection efficiency requires the acquisition
time to be increased by a factor of four to obtain the same signal-to-noise ratio. The detectors
of FCS instruments are therefore single-photon avalanche photodiodes (SPADs) or highefficiency PMTs with GaAsP cathodes [2].
The electronic setup of an FCS system is shown in Fig. 35. SPADs are connected via a cable
adapter (see Fig. 20) to the LVTTL Inputs of the DPC. Any SPAD module with TTL or
LVTTL output can be used. PMTs are connected to the CFD inputs, see Fig. 35, right.
DPC-230 module
SPADs
Detectors
H7422P-40
LVTTL
Inputs
Preamplifier
HFAC-26-2
DPC-230 module
H7422P-40
DCC-100 Detector Controller
Extension cables
id Quantique, MPD,
Perkin Elemer, Sensl
H7422 Power Supply and
Overload Shutdown
Fig. 35: FCS setup with DPC-230. Left: SPAD detectors. Right: GaAsP PMT modules
The measurement control part of the System parameters is shown in Fig. 36. ‘Operation
Mode’ is set to ‘Absolute Time’, ‘Correlation’. The settings shown on the left calculate and
display FCS curves, the accumulated number of counts, and an MCS (intensity) trace online.
The time-tag data are not saved. The settings shown on the right calculate the same curves, but
simultaneously save the time-tag data into a file ‘fluorescein-a.spc’. The maximum file size is
1000 Mb. The measurement stops when this file size is reached. The measurement can also be
26
Typical Applications
stopped after a defined acquisition time. Activate the ‘Stop T’ button if you want to stop after
a specified time.
‘Max Buffer Size’ defines a buffer in the RAM of the computer. If enough RAM is available
the buffer size should be as large as or larger than the specified file size. The SPCM software
then stores the time-tag data into the RAM before it writes them to the hard disc. The data
readout is then not slowed down by hard disc operations.
The experiment trigger is not used; the setting is ‘none’. You may use the experiment trigger
if you want to start the recording synchronously with an external event.
‘Collection time defines the acquisition time for the ‘Stop T option. ‘Display Time’ is the update interval for the online display.
Fig. 36: FCS: Measurement control part of the ‘System Parameters’ panel. Left without, right with saving of the
time-tag data.
The online display is configured via the configure panel shown in Fig. 37. With the settings
shown decay curves, FCS curves, and MCS (intensity) traces are calculated. FCS curves are
calculated by a multi-tau algorithm, and within a correlation time window of 1 ms. The MCS
traces are displayed at a resolution of 1 ms per point.
The SPCM software is able to run a fit on the calculated FCS data. The setting shown left are
without, the settings shown right with an FCS fit. The model contains two diffusion times,
tau 1 and tau 2, and a triplet time, Tau trip. F is the relative triplet population, N the number of
molecules in the detection volume, and Y the relative amplitude of the fast diffusion component. For the online fit we recommend to use a simple model, preferably one diffusion time
and a triplet time. The start values of the fit procedure are defined on the right of the ‘Configure’ panel.
Fig. 37: Configuration of the online display. Decay curves, FCS curves, and an MCS trace are displayed. The left
setup is without, the right setup with an FCS fit.
Fluorescence Correlation
27
The curves to be displayed in the display windows for the accumulated counts, FCS curves,
and MCS traces are defined under ‘Trace Parameters’. Each display window has its own trace
parameters. To open the trace parameters, click on ‘Display’, ‘Trace Parameters’. A click into
one of the curve windows selects the right trace parameter set. Alternatively, you can click in
the desired curve panel with the right mouse key, and select ‘Trace Parameters’. Examples of
the trace parameters are shown in Fig. 38. The trace parameters for the ‘Counts’ window activate the display of the accumulated counts for two detectors, connected to channel 4 and
channel 6. The trace parameters of the FCS window are set to calculate and display an FCS
curve for channel 4, and a fit to the data in channel 4. The trace parameters for the MCS window define traces for channel 4 and channel 6.
Fig. 38: Left to right: Trace parameters for Counts window, FCS window, and MCS window
A result of an FCS measurement with these parameters is shown in Fig. 39. The photons were
acquired by two detectors, at an average total count rate of 260,000 photons per seconds. The
total acquisition time was 46 seconds. The figure shows the accumulated counts of both detectors, the FCS (autocorrelation) of the detector connected to channel 4, and MCS traces for
both detectors.
Fig. 39: Result of an FCS measurement
Cross-Correlation between different detector channels is obtained by simply defining the
channels to be correlated in the trace parameters, see Fig. 40, left. The parameters shown correlate DPC channel 4 against channel 6, and run a fit on the calculated cross-FCS curve. The
result is shown in Fig. 40, right. The data points are shown pink, the fit with a single diffusion
time and a triplet time is shown black. The result of the fit is displayed by clicking on the
‘Trace Statistics’ button. They are shown as an insert in Fig. 40, right.
28
Typical Applications
Fig. 40: Cross-Correlation between two detector channels
Picosecond Fluorescence Correlation
Fluorescence correlation down to the picosecond range has first been described in [12]. The
authors used two bh SPC-130 modules with synchronised macro times, and included the TAC
times in the calculation. The time resolution of this system is extremely high. The timechannel width can be as small as 820 fs; the electronic IRF width is about 5 ps FWHM. The
resolution is therefore only limited by the transit-time spread of the detector. However, because the TAC times are not synchronous with the macro time clock the approach requires
relatively complicated data analysis [13]. Of course, detector dead time and afterpulsing preclude ps correlation to be obtained from a single detector. Therefore, at least two detectors
have to be used and the photon be data cross-correlated.
With the DPC-230, picosecond fluorescence correlation is merely a matter of the display scale
defined in the FCS display. A result is shown in Fig. 41. The trace parameters define two
traces, one for DPC channel 4 correlated versus channel 6, the other for channel 6 correlated
versus channel 4. The data points are shown red and black, respectively.
Fig. 41: Picosecond FCS. Left: Trace parameters. Right: FCS data. Time axis from 100 ps to 1 ms.
In contrast to classic FCS, in picosecond FCS transit time differences of the signals are noticeable. The result of such differences is a shift in the curves depending on whether detector
one is correlated against detector 2 or vice versa. Therefore, make sure that the optical path
lengths and the cable lengths are the same in all detector channels.
Anti-Bunching
29
Anti-Bunching
Anti-bunching information is contained in picosecond fluorescence correlation data, see Fig.
41. However, anti-bunching curves can also be recorded via the classic Hanbury-Brown-Twiss
start-stop experiment [14]. The light is split in two or more components and fed into separate
detectors. The photons of one component are used as start pulses, the photons of another detector as stop pulses. The photon pulses in the stop channels must be delayed in order to obtain positive start-stop times. A delay can be introduced by increasing the optical path length,
by increasing the cable length, or, in the DPC-230, by defining a TDC offset in the system
parameters. Except for the delay, the optical and the electrical setup are the same as for fluorescence correlation. The difference is only in the interpretation of the data. Picosecond FCS
calculates cross-correlation between different detectors, the classic start-stop experiment
builds up histograms of the times between the photons recorded in different detectors.
DPC system parameters for classic anti-bunching are shown in Fig. 42. In the simplest case,
the DPC builds up the start-stop histograms online, without saving any data of the individual
photons Fig. 42, left. However, the DPC can also build up FCS curves simultaneously with
the start-stop histograms (Fig. 42 middle and right). The single-photon data can be discarded
(Fig. 42, middle), or saved for later analysis (Fig. 42, middle).
Fig. 42: System parameters for classic start-stop experiments. Left: Histograms built up online, single-photon
data discarded. Middle: Histograms and FCS curves built up online, single photon data discarded. Right: Histograms and FCS curves built up online, single-photon data saved.
The input configuration panel is shown in Fig. 43. In the example shown two detectors are
used. The start detector is connected to channel 4 (LVTTL Input 2), and declared as ‘Reference’. The second detector is connected to channel 6 (LVTTL channel 4) and used as a stop
detector. More stop detectors can be used, and declared as ‘Input’ channels.
30
Typical Applications
Fig. 43: Input configuration for a classic anti-bunching start-stop experiment
To see the desired start-stop histograms, activate the traces for the input channels (i.e. the stop
channels) used in the trace parameters. The parameters for the two-detector setup defined in
the input configuration are shown in Fig. 44. A result obtained from a Rhodamine 110 solution is shown in Fig. 45.
Fig. 44: Trace parameters for a start-stop experiment, detector channel 6 against the detector declared as ‘reference’ in the input configuration.
Fig. 45: Anti-bunching curve obtained with the settings shown above
With the setups shown in Fig. 42, middle or right, classic anti-bunching histograms together
with correlation curves are obtained. A main panel with a start-stop histogram and an FCS
curve is shown in Fig. 46.
Fluorescence Lifetime Imaging
31
Fig. 46: Start-stop histogram and ps correlation obtained in the same measurement
Fluorescence Lifetime Imaging
The general principle of the TCSPC FLIM mode of the DPC-230 is the same as for the FIFO
Imaging mode of the bh SPC modules [2]. The DPC-230 records the photons detected in one
detector together with the synchronisation pulses from the scanner into a common time-tag
data stream. By analysing these data, the SPCM software assigns each individual photon to a
particular pixel of the scan area and to a particular time channel within this pixel, see Fig. 14,
page 11.
The basic system setup is illustrated in Fig. 47. The laser scanning microscope must either be
a multiphoton microscope (with a Ti:Sapphire laser) or a ps diode laser must be connected to
the scan head. The fluorescence light is coupled to the detector either from a non-descanned
port or a confocal output of the scan head. The single-photon pulses of the detector are fed
into CFD input 3 of the DPC module. A synchronisation signal from the laser is connected to
CFD input 4. For lasers with variable pulse period the SYNC pulses are delayed to make the
recording independent of the pulse period, see [2].
Laser Scanning Microscope
PMC-100
Detector
Ti:Sa or ps diode
laser
Scan
head
SYNC
from
Laser
DPC-230 module
Light
Light
LVTTL
Inputs
Scan
Clock
Pulses
DCC-100
Detector
Controller
Fig. 47: Basic system setup of a FLIM system
32
Typical Applications
The scan clock pulses (pixel clock, line clock, frame clock) of the microscope are connected
into three LVTTL channel of TDC 1. Thus, TDC 1 records the times of the scan clocks,
TDC 2 the times of the photons and the times of the laser pulses. The FLIM image is reconstructed from these data.
The system parameters for FLIM operation are shown in Fig. 48. The DPC works in the
‘TCSPC FIFO’ ‘FIFO Imaging’ mode. With the settings shown, a FLIM image with 256x256
pixels and 256 time channel per pixel is produced. Intermediate results are displayed in intervals of ‘Display time’. The image data are calculated online, the time-tag data are not saved. If
you need the time-tag data, e.g. for image correlation within a recording over a large number
of frames, activate the ‘Save .spc file’ button. No stop condition is set; the measurement runs
until it is stopped by the operator. If you want the measurement to stop after a specified ‘Collection Time’, activate the ‘Stop T’ button.
Fig. 48: System parameters for FLIM operation
The input configuration is shown in Fig. 49. TDC 1 works in the LVTTL mode. The inputs 3,
4, and 5 are defined as frame clock, line clock, and pixel clock inputs. TDC2 works in the
CFD input mode. CFD 3 receives the photon pulses, CFD 4 the laser reference pulses.
Fig. 49: Input configuration for FLIM operation
The details of the scan synchronisation are defined under ‘More Parameters’. In the simplest
case (i.e. for the bh DCS-120 scanner), the FLIM image is recorded at the same resolution the
microscope uses to scan the sample. The ‘More Parameters’ panel for this situation is shown
in Fig. 50, left. However, the DPC-230 can also apply line and pixel binning to the FLIM data.
Binning can be useful if a microscope scans at a pixel resolution higher than appropriate for
Fluorescence Lifetime Imaging
33
the FLIM image. Moreover, many microscopes send a frame clock pulse some pixels before
the start of the useful part of the line. These pixels can be excluded from the recording by defining a ‘Left Boarder’ larger than zero. Settings typical for FLIM with the Zeiss LSM 510 and
LSM 710 microscopes [7] are shown in Fig. 50, right.
Fig. 50: Scan synchronisation parameters. Left: FLIM image has same pixel numbers as microscope scan. Right:
Pixels and lines are binned by a factor of two, and the recording starts 11 pixels after the line clock.
The recommended main panel configuration for DPC-230 FLIM with the bh DCS-120 scanner [4] is shown in Fig. 51. A time-gated image is displayed on the left, the scanner control
panel is kept open on the right.
Fig. 51: Recommended main panel configuration for FLIM. Gated Intensity image and scan parameters of
DCS-120 system displayed.
A measurement in the FLIM modes of the DPC module delivers the photon distribution over
the coordinates of the scan and the time within the fluorescence decay. The data can be considered an array of pixels, each containing a large number of time channels spread over the
fluorescence decay. To obtain fluorescence lifetimes the decay curves in the individual pixels
must be fitted with an appropriate model. The SPCM software has the SPCImage [4] FLIM
data analysis integrated. A lifetime image obtained by fitting a triple-exponential model to the
data is shown in Fig. 52.
34
Typical Applications
Fig. 52: Fluorescence lifetime image recorded with DPC-230 and bh DCS-120 confocal scanning FLIM system
[4]. Data analysed by SPCImage data analysis routines of SPCM software [4, 6]. Intensity image, lifetime image,
decay curve and fit at cursor position.
Luminescence Lifetime Imaging in the Microsecond Range
Fluorescence lifetime imaging in the microsecond range requires low repetition rate of the
excitation pulses. Low repetition rate causes two problems. The first one is that the laser repetition rate interferes with the pixel rate. Unless extremely slow pixel rates are used the result is
stripes in the image. Second, the ratio of peak power to average power becomes large. Especially if femtosecond or picosecond lasers are used the high peak power may cause multiphoton excitation and excited-state absorption. If the peak power is reduced the average
power becomes low, resulting in low emission intensity and long acquisition time.
The solution is to use excitation pulses of nanosecond or microsecond duration, and to synchronise these pulses with the pixel clock of the scanner. The principle is shown in Fig. 53.
Laser Scanning Microscope
BDL-SMC Diode Laser
in CW mode
Scan
head
PMC-100
Detector
SYNC
from
Pulse
Generator
DPC-230 module
Light
LVTTL
Inputs
Scan
Clock
Pulses
CW
ON/
Off
Power
DCC-100
Detector
SYNC
DDG-200 card
or other
Pulse generator
Controller
Pxl Clock
Trigger
Fig. 53: Microsecond FLIM. The laser pulses are synchronised with the pixel clock.
The general setup is the same as for FLIM in the nanosecond range, compare Fig. 47. However, the BDL-SMC laser is operated in the CW mode [5] and controlled by a bh DDG-200
card or another pulse generator. The laser is switched on for a specified time interval at the
Luminescence Lifetime Imaging in the Microsecond Range
35
beginning of each pixel. The pixel time is made long enough to observe the full luminescence
decay before the scanner goes to the next pixel.
Because of the low laser repetition rate the DPC-230 is best operated in the ‘Multiscaler Imaging’ mode. This makes a delay in the SNC path unnecessary. Moreover, multiscaler operation
does not require any gating of the stop pulse with the previously detected photons (see Fig. 4).
Therefore, 12 LVTTL input channels (16 minus 4 for the scan clocks and the SYNC) are
available to connect detectors. An example of a system with several SPADs is shown in Fig.
54.
Laser Scanning Microscope
BDL-SMC Diode Laser
in CW mode
SPAD
Detectors
Scan
head
DPC-230 module
Light
Scan
Clock
Pulses
CW
ON/
Off
SYNC
from
Pulse
Generator
LVTTL
Inputs
Power
DCC-100
Detector
SYNC
DDG-200 card
or other
Pulse generator
Controller
Pxl Clock
Trigger
Fig. 54: Microsecond FLIM system with SPAD detectors
System parameters for a microsecond FLIM system are shown in Fig. 55. The operation mode
is ‘Multiscaler, ‘FIFO Image’. An image of 256 x 256 pixels is acquired; the acquisition runs
until the measurement is stopped by the operator. Each pixel contains 256 time channels, with
a width of 39 ns. The total time range covered is about 10 µs. The image is built up online; the
time-tag data are not saved. If you want to save the data, e.g. for running image correlation on
data acquired over many frames, activate the ‘Save .spc file’ button.
Fig. 55: DPC System parameters for microsecond FLIM
The input configuration panel is shown in Fig. 56. The configuration for a setup as shown in
Fig. 53 is shown left. The PMT is on CFD 3, the reference on CFD 4. The scan clock pulses
are connected to LVTTL 1, 2, and 3.
The input configuration panel for the setup shown in Fig. 54 is shown right. The reference is
on LVTTL 4, LVTTL 5 through LVTTL 20 record signals from SPADs.
36
Typical Applications
Fig. 56: Input configuration panel. Left: PMT on CFD 3, reference on CFD 4. Right: SPADs on LVTTL 5
through 20, reference on LVTTL 4.
Fig. 57 shows a luminescence lifetime image of a luminescent glass embedded in a Rhodamin 6G solution. The image was recorded by a bh DCS-120 confocal scanning FLIM system
[6] and analysed by the bh SPCImage routines embedded in the SPCM software. The glass
fluorescence has a triple-exponential decay, with components of 140 ns, 689 ns, and 1,79 µs.
Fig. 57: Microsecond FLIM, analysed with SPCImage. Fluorescent glass (left) embedded in Rhodamin 6G solution (right). Colour represents lifetime. Triple-exponential decay analysis, decay components of glass fluorescence 140 ns, 689 ns, and 1,79 µs. bh DCS-120 confocal scanning FLIM system with bh BDL-405SMC laser.
SPCM Software
The DPC-230 comes with the ‘Multi SPC Software’, or ‘SPCM’ operating software. The
SPCM software is not only used for the DPC-230 but also for all bh SPC (TCSPC) modules.
It allows the user to operate up to four DPC-230 modules, or up to four SPC-630, -730, -830,
130, 140, or 150 modules. The SPCM software includes measurement parameter setting,
measurement control, loading and saving of measurement and setup data, and data display and
evaluation in 2-dimensional and 3-dimensional modes. The SPCM software runs under Windows 2000, NT, XP, and Vista. For installation of the SPCM software, please see
‘Installation’ page 13. The SPCM software is described in detail in the ‘bh TCSPC Handbook’
[2]. The following chapter focuses mainly on the features which are specific of the DPC-230.
Configuring the SPCM Main Panel
The main panel of the SPCM software can be adjusted to different instrument configurations.
For details please see [2], ‘SPCM Software’. There may be several windows for the measurement results, or different results may be displayed within one window. Frequently used control panels may be kept open at any time. Some typical configurations are shown in Fig. 58.
Fig. 58: Different configurations of the main panel. Upper row, left: Absolute time mode, with numbers of photons accumulated, intensity trace, cross-correlation of two channels, and photon counting histograms. Upper row,
right: TCSPC measurement. Lower row, left: TCSPC FIFO measurement. Fluorescence decay and FCS curve.
Lower row, right: Measurement in the multichannel scaler mode. Waveforms recorded into different measurement pages.
38
SPCM Software
A typical main panel of a correlation measurement in the ‘Absolute Time’ mode is shown in
the upper row, left. It shows the photon numbers accumulated in four detection channels, the
intensity trace of one channel, correlation between two channels, and photon counting histograms of two channels.
A TCSPC measurement is shown in the upper row, right. It shows a fluorescence decay curve
and an IRF recording. The recordings were taken in different ‘Measurement Pages’. The panels of the ‘display parameters’ and the ‘trace parameters’ are kept open on the right.
The lower row, left, shows a combined decay/FCS measurement in the TCSPC FIFO mode. It
shows the decay curve, the intensity trace, and the FCS curve for one detector.
A measurement in the Multichannel Scaler mode is shown in the lower row, right. Two subsequent measurements were recorded into subsequent ‘pages’ of the memory, and are displayed simultaneously.
Pleas note that the setup parameters for frequently configurations can be put in a list of ‘Predefined Setups’, see ‘Predefined Setups’, page 51. Changing between different instrument
configurations is then a matter of a single mouse click, see below.
Changing Between Different Instrument Configurations
The complete instrument configuration is saved together with the data or setup files [2]. Thus,
the main panel configuration used for a particular experiment is restored when the corresponding measurement or setup data are loaded. Moreover, you may put frequently used system
configurations into a list of ‘predefined setups’, see page 51. By using the panel shown in Fig.
59 you can change between different instrument configurations by a single mouse click.
Fig. 59: Predefined-Setup panel. You can change between different instrument configurations by a single mouse
click
Display and Trace Parameters
The display windows of the main panel are configured by the ‘Display Parameters’ the ‘2D
Trace Parameters’, and the ‘3D Trace Parameters’. The corresponding panels are shown in
Fig. 60.
Fig. 60: Left to right: Display parameters, 2D Trace parameters, and 3D Trace Parameters
Configuring the SPCM Main Panel
39
The Display Parameters (shown left) allow you to define how your results will be displayed.
The upper part refers to the display of curves, the lower part to the display of images or other
multi-dimensional data. The range of the count numbers to be displayed can be defined in the
upper left. The style of the curves displayed can be defined under ‘Trace’ and ‘2D display’.
The colours of images are defined in the lower left. A 3D curve mode, a colour-intensity
mode, and an OGL plot are available. Sub-sets of multi-dimensional data sets, e.g. images in
different time or wavelength windows, can be selected in the lower right part of the panel.
Examples are given in section. For details, please see ‘The bh TCSPC Handbook’ [2]..
As shown in Fig. 58, the main panel may have several display windows open. The windows
have separate Display and Trace Parameters. To select the right set of display parameters,
click into the top bar of the particular display window, or click into the desired display window with the right mouse key.
The 2D Trace Parameters define the curves displayed 2-dimensional display modes. Curves
from different modules of a multi-module TCSPC system (‘Module’), individual curves from
a multi-detector measurement (‘Curve’), and from different measurements or steps of a measurement sequence (‘Frame’ or ‘Page’) can be selected. A curve may also contain accumulated
data of several detector channels or measurement steps.
The 3D Display Parameters define up to eight display windows for multi-dimensional measurements. For each window, the display mode can be defined individually. Thus, images in
defined time windows, sequences of curves in defined windows of an image, or a defined windows of the detector number can be defined. Each window has separate display parameters.
Please see also [2].
Resizing and Positioning the Display Windows
To make the windows of the main panel resizable click into ‘Display’ and select ‘Scale Contents on Resize’. To resize a window, seize the edge of the window with the mouse cursor and
pull the panel to the desired size. To shift a window, seize the top bar of the window and shift
it into the desired position, see Fig. 61.
Fig. 61: Resizing and shifting windows of the main panel
Clicking into a display window area by the right mouse key opens the select panel shown in
Fig. 62. ‘Proportional Graph’ sets the display proportions according to the ‘Scan Pixels X’ and
‘Scan Pixels Y’ of a Scan measurement. ‘Full Size Graph’ spreads the display window over
the maximum available area. The panel also allows you to enable or disable the cursors, and to
access the Display Parameters and Trace Parameters.
40
SPCM Software
Fig. 62: Select panel for display size, cursor display, and display and trace parameters
It can happen that a display window has disappeared behind the edge of the screen or otherwise got out of control. (This can happen if a file from a dual-screen system is loaded in a
computer with only one screen.) In that case, click into ‘Display’, and ‘Default Size and Position’. When the window is back in the screen area, set it to ‘Scale Contents on Resize’, see
Fig. 63.
Fig. 63: Left: Setting window sizes and positions to default. Right: Window sizes user-definable
Cursors in the Display Windows of the Main Panel
Cursors in the display windows of the main panel are enabled by clicking into the window
with the right mouse key. This opens a small panel in which the cursor functions can be enabled or disabled, see Fig. 64, left. A display window with cursors is shown in Fig. 64, middle. Two cursors and a ‘data point’ are available.
When the cursors are enabled a window with the cursor settings is displayed, see Fig. 64,
right. The cursor settings window can be placed anywhere in the screen area. It can be closed
by clicking on the ‘close’ symbol in the upper right corner, and re-opened by a right mouse
click into the display window and selecting ‘Cursor Settings’.
Fig. 64: Cursors in the display windows of the main panel
The cursors and the data point can be shifted by the mouse cursor, or by changing the cursor
positions in the cursor settings window. Furthermore, the style and the colour of the cursors
can be changed and a zoom function is available. The cursors in the main window interact
with the parameters of the bh DCS-120 confocal scanning system, see [6].
Link to Data Analysis
The SPCM data acquisition software has a direct link to the SPCImage data analysis. Data are
sent to the data analysis by clicking on ‘Main’, ‘Send Data to SPCImage’. For a number of
Configuring the SPCM Main Panel
41
instrument configurations several data sets may have been recorded. In this case click on the
display window that shows the data to be analysed, and then click on ‘Send Data to SPCImage’, see Fig. 65. For details of the SPCImage data analysis, please see [4] and [6].
Fig. 65: Sending data to the data analysis. Click on data set to be sent (left), click on ‘send data to SPCImage’
(middle), SPCImage opens with the data selected (right)
Trace Statistics
Clicking on the ’Trace Statistics’ button opens a window which displays information about the
data shown in the curve windows. For decay curves or other waveforms the FWHM values,
the peak counts, the total counts and the first moment of the photon distribution are displayed,
see Fig. 66, left. For an FCS window the trace statistics window displays the results of the fit,
see Fig. 66, right.
Fig. 66: Trace statistics panel. Left for a waveform window, right for FCS window
The window can be placed anywhere in the screen area. Please note that the trace statistics
window works also in the oscilloscope mode. The window is thus an efficient tool to adjust
the system for best time resolution, correct the signal transit time, and optimise the counting
efficiency or IRF stability.
Status Information
Count Rate Display
The count rate display informs about the count rates of the two
TDCs of the DPC-230. Both rates are the total rates for all active
channels of the TDCs. Important: In contrast to the bh SPC modules the TDC chips have no counters to determine the count rates
directly. The count rates have therefore to be determined by the
SPCM software from the incoming photon data stream. Because
the photon data of each TDC are buffered in FIFO there may be
some latency in the count rate display.
Fig. 67: Count Rate Display
FIFO Status
The status of the on-board FIFO buffers are displayed in the ‘Device State’ window. The filling of the buffers is shown by ‘FIFO
Usage’. A FIFO overflow is shown by the indicator on top of the
FIFO usage bars. When an overflow occurs there is a gap in the
recorded photon data stream, and the absolute time scale may be
Fig. 68: FIFO status
42
SPCM Software
lost. Strictly, such data cannot be correlated any more. However, as long as only a few overflows occurred correlation within one and the same TDC may still possible without noticeable
errors. However, data with more than 10 overflows are useless for correlation.
Device State
’Device state’ informs about the general status of the DPC-230 see Fig. 69.
Fig. 69: Device status information
The most common status messages are
Collecting Data:
User break:
Displaying Data:
Displaying data from file:
Total 275635 events collected:
Displaying data from file:
Overflow(s) occurred:
Waiting for Trigger:
Measurement running
Measurement has been stopped by the operator
Measurement finished, final result is displayed
Data loaded from an .sdt file are displayed
Measurement has recorded the indicated number of events
Displaying data loaded from an .sdt data file
The FIFO has run full, not all photons could be recorded
Measurement started, waiting for experiment trigger
System Parameters of the DPC-230
43
System Parameters of the DPC-230
The system parameters panel of the DPC-230 is shown in Fig. 70. The panel contains separate
sections for the measurement control parameters and the hardware settings of the CFDs and
the TDCs.
Fig. 70: System parameters of the DPC-230
Operation Mode
The operation mode of the DPC is selected by two parameters. The first parameter determines
the hardware configuration, the second one the way the photon data are interpreted. The selection panel is shown in Fig. 71.
Fig. 71: Selection of the operation mode
Absolute Time
‘Absolute Time’ is an absolute timing mode. It records the photons of all active channels (see
‘Absolute Timing’, page 2) with their times from the start of the experiment. The way of interpretation is ‘Correlation’. The Absolute Time mode is used to obtain intensity traces, photon counting histograms, and fluorescence correlation and cross-correlation functions for a
large number of detector channels. Correlation can be obtained over a wide time range from
picoseconds to seconds.
TCSPC FIFO
‘TCSPC FIFO’ records three detector signals and one reference signal via the CFD inputs.
The mode delivers both relative and absolute photon times. The absolute times are referred to
the start of the measurement, the relative times to reference pulses from the light source. The
time measurement is ‘reversed start-stop’, i.e. the times are measured from the photons to the
44
SPCM Software
next reference pulse (see ‘Relative Timing’, page 3). The data of the detector channels can be
interpreted either as single waveforms (‘Single’), as oscilloscope traces (‘Oscilloscope’), or as
fluorescence decay curves plus fluorescence correlation data (‘FIFO’). The ‘FIFO’ option is
similar to the FIFO mode of the bh SPC modules [2]. Moreover, images can be acquired by
recording synchronisation pulses from a scanner together with the photons (‘FIFO Image’).
Please note that ‘TCSPC’ is available only for the CFD inputs.
Multichannel Scaler
The ‘Multiscaler’ mode records both relative and absolute photon times. The times are referred to a reference pulse from a light source (see ‘Multichannel Scaler Mode’, page 8). In
contrast to ‘TCSPC FIFO’ every reference pulse is rescored, not only the next one after the
detection of a photon. The times are measured from the reference pulses to the photons. The
‘Single’ option accumulates the photon density over the time after the reference pulses, i.e.
builds up the waveform of the light signals. The ‘Oscilloscope’ mode runs a repetitive measurement and displays subsequent waveforms in short intervals. ‘FIFO’ is used when fluorescence correlation in combination with photon density histograms is required. Images can be
acquired by recording synchronisation pulses from a scanner together with the photons (‘FIFO
Image’). The ‘Multiscaler’ function is available both for the CFD inputs and for the LVTTL
inputs.
Specification of the Time-Tag Date File
The DPC-230 records time-tagged data, i.e. the times and TDC channel numbers of the individual photons. From these data can be calculated waveforms, intensity traces, correlation
curves, or images. Because these data operations are performed online on the incoming data
stream the time-tag data need not necessarily be saved in a file. However, it may be useful to
save the time-tag data, e.g. to be able to re-analyse the data later, or to import them into other
analysis software. Therefore, the time-tag data can be stored in two data files, one for TDC 1,
the other for TDC 2. For data format please see ‘Format of Time-Tag Data Files’, page 55.
The corresponding control section of the system parameters is shown in Fig. 72.
Fig. 72: Options for saving the raw data
The time-tag data recorded by the DPC-230 can easily reach sizes of tens or hundreds of
megabytes. The maximum used disk space can therefore be limited by activating the ‘Limit
Disk Space’ button and specifying the maximum file size. The measurement stops when the
specified disk space has been filled.
‘Maximum Buffer Size’ defines a buffer in the computer memory. The buffer stores the data
before they are saved to the hard disk. If possible, the buffer should be large enough to buffer
the FIFO data of the complete measurement. Usually, the buffer size can be as large as
250 Mb for a computer with 1 Gb RAM. Under no circumstances a buffer size larger than the
available memory space of the computer should be used. Windows may then attempt to provide virtual memory, i.e. to swap memory space with the hard disc. This makes the computer
extremely slow and almost certainly causes problems with the data transfer from the DPC
module.
If a sufficiently large buffer is not available use a buffer smaller than the value specified under
‘limit disk space’. Of course, the SPCM software writes to the hard disk when the buffer is
System Parameters of the DPC-230
45
full. Although this may slow down the data transfer from the SPC module the loss is by far
smaller than for memory swapping.
You may switch off the storing of the time-tag data altogether. However, in this case the software discards the raw data and delivers (and stores) only the results specified under ‘Configure’ and. It is then impossible to run any later data processing on the single-photon data. The
option should therefore be used with care.
Name of Time-Tag Data File
During the measurement, the time-tag data are stored in a temporary file. The final data file is
written when the measurement has been completed. The name of the time tag file time-tag
data file is therefore specified at the end of the measurement. The definition panel is shown in
Fig. 73. A list of previously used data file names is available by clicking on the symbol.
Fig. 73: Definition of the name of the time-tag data files
Configuring the Runtime Display
The SPCM software runs online processing on the incoming DPC data. The functions to be
calculated on-line are shown in the field under ‘Run Time Display’. The run time display can
be configured by clicking on the ‘Configure’ button. This opens the panel shown in Fig. 74.
Fig. 74: Configuration panel for the runtime display. Left without, right with fitting of FCS data
The available on-line functions are
-
Calculation of decay curves (photon density distributions) for the individual detectors
FCS by a linear-τ algorithm with subsequent binning or FCS by a multi-τ algorithm. The
maximum time up to which the correlation is calculated is defined by ‘Correlation Time’.
Fluorescence cross correlation between different detectors. (Use the 2D Trace parameters
to define the detector channels)
46
-
SPCM Software
Calculation of photon counting histograms for the individual detectors (‘FIDA’). The
sampling time interval is specified on the right.
Calculation of lifetime histograms (FILDA) for all detectors. The sampling time interval is
specified on the right.
Intensity traces (MCS) for the individual detectors.
For all functions specified for runtime display individual display windows are provided in the
main panel, see ‘Configuring the SPCM Main Panel’ page 37. The data displayed within these
windows are defined by the ‘Trace Parameters’. Each display window has its own set of trace
parameters. The trace parameter definitions for ‘Decay’, ‘FIDA’, ‘FILDA’ online display are
shown in Fig. 75. Traces for DPC channels 1 to 4 are displayed.
Fig. 75: Trace Parameters of the DPC-230, for decay curve, PCH, and MCS
The trace parameters for ‘FCS’ are shown in Fig. 76. The trance parameters for FCS without a
fit are shown left. ‘Channel’ and ‘Cross FCS Channel’ define the DPC detector channel numbers for the data to be correlated and displayed. If ‘Curve’ and ‘Cross-FCS Curve’ are the
same the autocorrelation function of the channel is calculated. Thus, the configuration shown
calculates the autocorrelation functions of channel 4 and 5, and the cross-correlation of channel 4 versus channel 5.
If the fit function in the runtime configuration panel is enabled (see Fig. 74, right) the trace
parameter panel is as shown in Fig. 76, right. For each trace can be defined whether an FSC
curve or a fit curve is displayed.
Fig. 76: Trace parameters for FCS display. Left without, right with fit of the FCS curves
For reasons of computation speed and memory space, the software calculates only one correlation function per detector channel. If the Trace Parameters define several correlation functions
for one channel, e.g. an autocorrelation and a cross-correlation with another channel, only the
first correlation is calculated during the measurement. The other ones are calculated and displayed after the measurement has been completed.
Configuring the Inputs
The configuration panel for the configuration of the DPC inputs is shown in Fig. 77. The
panel is opened by clicking on ‘Input Configuration’ in the system parameter panel.
System Parameters of the DPC-230
47
Both TDC chips can be switched on and off by the ‘active’ button. If one of the TDCs is not
used it should be switched off to avoid unnecessary software actions.
For both TDCs the inputs can be switched to ‘LVTTL’ or ‘CFD’. Each TDC either has 8
LVTTL inputs or 2 CFD inputs. The LVTTL configuration is shown in Fig. 77, left, the CFD
configuration right. It is possible to combine LVTTL operation in one TDC with CFD operation in the other.
Fig. 77: Input configuration panel
All inputs can be individually switched on and off by the ‘active’ buttons. Moreover, the function of the inputs can be defined. The function can either be an input for a detector signal, a
reference input from a light source, or a marker input. For TCSPC operation channel 12
(CFD 4) is always the reference.
‘Edge’ defines the active edge of the input signal. The active edge of the CFD inputs is always
the falling edge; for the LVTTL inputs either the rising or the falling edge may be used.
Please note: For SPAD modules with TTL outputs ‘rising edge’ must be selected.
CFD and SYNC Parameters
The CFD parameters define the thresholds and the zero-cross levels of the CFD inputs. The
thresholds can be set from 0 to -510 mV, the zero-cross levels from -96 to +96 mV. CFD
thresholds smaller than -20 mV can result in noise pickup and should therefore be avoided. If
a detector requires a threshold this low a preamplifier of sufficient gain should be used.
Fig. 78: CFD threshold and zero cross settings.
Please note that in the TCSPC mode CFD 4 is used for the reference input.
SYNC Frequency Divider
In the TCSPC mode the fourth CFD channel is used as a reference input. To display either 1,
2, or 4 signal periods of the recorded signal (see Fig. 6, page 4) the fourth CFD channel has a
selectable frequency divider. The divider ratio is selected under ‘SYNC parameters’. The parameter is available only in the TCSPC mode.
48
SPCM Software
Fig. 79: SYNC frequency divider setting. The parameters determines the number of signal periods recorded in the
TCSPC mode
TDC Parameters
The TDC parameters determine the recorded time interval and the number of time channels in
the TCSPC mode and in the multichannel-scaler mode. The parameters are shown in Fig. 80.
Fig. 80: Left: TDC parameters. Right: Selecting the number of time channels from a list
‘Range’ is the recorded time interval. ‘Offset’ provides a shift of the results in time. ‘Channel
Number’ is the number of time channels. Values from 1 to 16384 can be selected from a list,
see Fig. 80, right.
Time/Channel is either the time channel width of the TDC (164.6 ps) or a multiple of this
value. Time/channel is set automatically depending on the selected ‘Range’ and ‘Channel
Number’.
Because ‘Range’ must by a multiple of the time-channel width of the TDC the parameters
‘Range’ and ‘Channel Number’ are automatically corrected to the nearest possible combination. If the selected number of channels would result in a ‘Range’ larger than selected the
value for ‘Range’ is automatically increased. If the desired ‘Range’ cannot be reached with the
selected number of time channels the width of the time-channels is increased, i.e. several TDC
channels are binned into one wider channel.
The ‘Multiphoton Detection’ button defines whether only one or several photons per signal
period are recorded in the TCSPC mode. Please be cautious when using TCSPC with multiphoton detection. Even when multiphoton detection is enabled a second photon cannot be
detected within the dead time of the detector. Multiphoton detection therefore does not necessarily remove curve distortions by pile-up effects. On the contrary, dead-time effects can make
distortions even stronger, and less predictable than for single-photon detection.
Saving Setup and Measurement Data
49
Saving Setup and Measurement Data
The ‘Save’ panel is shown in Fig. 81. It contains fields to select different file types, to select
or specify a file, to display information about existing file, and to select between different save
options.
Fig. 81: Save panel
File Format
You can chose between ‘SPC Data’ and ‘SPC Setup’. The selection refers to different file
types. With ‘SPC Data’ files are created which contain both measurement data and system
parameters. When this file is loaded not only the measurement data are restored but also the
complete system setup. With ‘SPC Setup’ files are created that contain the system parameters
only. When such files are loaded the system setup is restored, but no data are loaded. Files
created by ‘SPC Data’ have the extension ‘.sdt’, files created by ‘SPC Setup’ have the extension ‘.set’.
File Name / Select File
A file name can be written into the ‘File Name’ field. ‘Select File’ opens a dialog box that
allows you to change or create directories. Moreover, it shows the names of existing files.
These are ‘.sdt’ files or ‘.set’ files, depending on the selected file format. If you want to overwrite an existing file you can select it in the ‘File Name’ field. A history of previously saved
files is available by clicking on the button.
File Info
After selecting the file text can be written into the ‘Author’, ‘Company’ and ‘Contents’ fields.
Both for ‘SPC data’ and ‘SPC setup’ the file information is saved in the file. The file information helps considerably to later identify a particular measurement among a large number of
data files. We therefore strongly recommend to spend a few seconds on typing in a reasonable
file information.
If you have selected an existing file the file information contained in it is displayed in the
‘File info window’. If you want to overwrite this file you can edit the existing file information.
Selecting the data to be saved
Under ‘What to Save’ the options ‘All used data sets’, ‘Only measured data sets’ or ‘Selected
data blocks’ are available, see Fig. 82. The default setting is ‘All used data sets’, which saves
all valid data available in the memory of the SPC modules. These can be measured data, cal-
50
SPCM Software
culated data or data loaded from another file. Except for special cases (see[2]) we recommend
to use the ‘All used data sets’ option.
Fig. 82: Save options
Loading Setup and Measurement Data
The ‘Load’ menu is shown in Fig. 83. It contains fields to select different file types, to specify
a file, to display information about the file selected, and to select different load options.
Fig. 83: Load panel
File Format
You can chose between ‘SPC Data’ and ‘SPC Setup’. The selection refers to different file
types. With ‘SPC Data’, .sdt files are loaded. These files contain both measurement data and
system parameters. Thus the load operation restores the complete system state as it was in the
moment when the file was saved.
If you chose ‘SPC Setup’, .set files are loaded. These files contain the system parameters only.
The load operation sets the system parameters, but the actual measurement data are not influenced.
Note: Measurements in the ‘FIFO’ (time tag) mode deliver an .spc file that contains the micro
time, the macro time, and the detector channel for each individual photon. These files are
loaded by using the ‘Convert’ routines, see [2].
File Name / Select File
The file to be loaded can is selected in ‘File Name’ field. ‘Select File’ opens a dialog box that
displays the available files. These are ‘.sdt’ files or ‘.set’ files depending on the selected file
format. A history of previously loaded files is available by clicking on the button.
File Info
The file info window displays information about the file selected. The first three lines of the
file info are inserted automatically when a file is saved. The last three items can be typed in by
the operator, see ‘Saving Setup and Measurement Data’.
Predefined Setups
51
Block Info
Activating a data block in the ‘Block Number in File’ field enables a ‘Block Info Button’.
Clicking on this button opens a list that contains the device number of the SPC modules by
which the data were recorded, the time and data of the recording, and all system parameters,
see Fig. 84. At the end of the block information the minimum and maximum count rates of the
corresponding measurement are shown (see Fig. 84, right). The block info often helps to recover the exact recording conditions of an older measurement.
Fig. 84: Block info window of the load panel
Load Options
Under ‘What to Load’ the options ‘All data blocks & setup’, ‘Selected data blocks without
setup’ or ‘Setup only’ are available. The default setting is ‘All data blocks & setup’, which
loads the complete information from a previously saved data file. Except for special cases
(see[2]) we recommend to use the ‘All data blocks & setup’ option.
Predefined Setups
Setups of frequently used system configurations can be added to a list of ‘predefined setups’.
Changing between these configurations then requires only a mouse click.
To use the predefined setup option, click on ‘Main’, ‘Load
Predefined Setups’. This opens the panel shown right. A
setup is loaded by clicking on the button left of the name of
the setup.
To add or delete setups to or from the list, or to change the
names of the setups, click into one of the name fields with
the right mouse key. This opens the panel shown in Fig. 85.
To add a setup, click on the disc symbol right of the ‘File
Name’ field and select a ‘.set’ file. Default setups coming with the SPCM software are in the
‘default setups’ folder of the working directory defined during the software installation. Please
note that there may by sub-directories for different classes of applications. Select the files you
want to put into the list of predefined setups, and click on the ‘Add’ button. Every setup has a
user-defined ‘nickname’. The default nickname is the file name of the .set file. To change the
nickname, click into the nickname filed and edit the name. Then click on ‘Replace’.
52
SPCM Software
Fig. 85: Editing the list of predefined setups
To create your own predefined setups, first save a setup file of the system configuration you
want to add the list. Use the ‘Save’ panel, option ‘setup’, as described under ‘Save’. Then add
the file to the setup list as described above.
You can also add an ‘.sdt’ file to the setup list. The .sdt file contains not only the system settings but also measurement data. You can define whether the file is loaded with or without the
data by clicking on the ‘load with data’ marker.
Importing FIFO Files
53
Importing FIFO Files
Measurements in the absolute time modes deliver an .spc file that contains time-tag data, i.e.
the micro time, the macro time, and the detector channel for each individual photon [2]. During a FIFO measurement normally decay curves, FCS curves, or photon counting histograms
are calculated on-line. These data are saved in normal .sdt files. However, it may happen that
not all of the desired functions were calculated during the measurement, or that the calculation
was not done with the optimal binning parameters or sampling time intervals. Moreover, online data analysis may have not been used altogether to increase the data throughput rate. In
these cases, the ‘Convert FIFO File’ function allows you to read the data from the .spc file
created during the measurement and calculate the desired correlation functions or histograms.
The Convert FIFO panel is shown in Fig. 86.
Fig. 86: Conversion of FIFO data into .sdt data
Each measurement creates a setup (.set) file which has the same name as the .spc file (or the
last .spc file if several are recorded sequentially). The name of this setup file must be specified
under ‘Setup File name’. As in the load and save panels, the file is specified in file name field.
Clicking on the disc symbol opens a dialog box that displays the available files. A history of
previously used setup files is available by clicking on the
button. The ‘File Info’ displays
information about the corresponding measurement.
The FIFO (.spc) file is specified under ‘Source File Name’. A FIFO measurement may produce several .spc files. The data of all these files are combined by activating the ‘Use Stream
of Files’ button. ‘Overall Measurement Time’ informs about the total time over which the
measurement has been run.
The lower part specifies the .sdt file to be created. The file name is specified under ‘Destination file name’. The conversion routine suggests a file with the same name as the .spc file.
54
SPCM Software
The ‘Convert FIFO’ routine allows you to convert .spc files into different destination file
types. The destination file type is specified in the ‘File Format’ field in the lower part of the
Convert panel. The most frequently used conversion is into .sdt files of the TCSPC FIFO
mode, see Fig. 87.
Fig. 87: Conversion of .spc files into .sdt files of the FIFO mode. Left: Configuration of destination data. Right:
Selection of the functions to be calculated and calculation parameters.
The functions to be calculated are defined in the ‘Select & configure histograms’ field. A click
into this field opens the panel shown in Fig. 87, right. It is the same panel used to configure
the online display of the FIFO mode, see ‘Configuring the Runtime Display’, page 45. You
can specify the calculation of decay curves, FCS functions, photon counting histograms
(FIDA), photon counting lifetime histograms (FILDA), and FCS traces.
A FIFO measurement may have been run over a long acquisition time and contain data from
several routing channels (detectors). Therefore, parameters are provided to control the structure of the calculated data. With ‘Time Interval’ and ‘Starting from Time’ a time interval
within the ‘Overall measurement time’ of the FIFO measurement can be selected. Moreover,
if the .spc data were recorded in several detector channels the full routing information may be
used, only a specified range of channels may be converted, or the routing information may be
ignored altogether, i.e. all detector channels merged.
Format of Time-Tag Data Files
55
Format of Time-Tag Data Files
In most of the operation modes the DPC-230 allows the user to record time-tag data of the
individual photons. The format of the time-tag data files is described in this section.
Time-tag data files consists of a sequence of 32 bit records. The first record is a descriptor that
identifies the file as a DPC-230 time-tag file and contains the time-channel width of the recording. The subsequent records contains photon data. Each record contains four bytes as
shown in Table 1.
1st Record = Descriptor
Byte 0
Bits [7:0]
Byte 1
Bits [15:8]
Byte 2
Bits [23:16]
Byte 3
Bits [31:24]
2nd Record
Byte 0
Bits [7:0]
Byte 1
Bits [15:8]
Byte 2
Bits [23:16]
Byte 3
Bits [31:24]
Table 1: Byte order in the .spc files
Descriptor
The structure of the descriptor is shown in Table 2.
Bit 31
Bit 30
Bit 29
Bit 28
Bit 27
Bit 26
Bit 25
Bit 24
1
1
0
0
0
RAW
0
1
Bit 23
Bit 22
Bit 21
Bit 20
Bit 19
Bit 18
Bit 17
Bit 16
TPB[23]
TPB[22]
TPB[21]
TPB[20]
TPB[19]
TPB[18]
TPB[17]
TPB[16]
Bit 15
Bit 14
Bit 13
Bit 12
Bit 11
Bit 10
Bit 9
Bit 8
TPB[15]
TPB[14]
TPB[13]
TPB[12]
TPB[11]
TPB[10]
TPB[9]
TPB[8]
Bit 7
Bit 6
Bit 5
Bit 4
Bit 3
Bit 2
Bit 1
Bit 0
TPB[7]
TPB[6]
TPB[5]
TPB[4]
TPB[3]
TPB[2]
TPB[1]
TPB[0]
Table 2: Format of the descriptor
RAW Bit
The RAW bit identifies whether or not the data in the file are pre-processed. ‘Raw = 1’ identifies data as they are read from the incoming data stream. Such data are difficult to use for several reasons: The TDC channels of the DPC board have individual FIFOs. The software may
therefore read the individual photons not in the order they were detected. Moreover, the internal clock of the TDC channels overflows in regular intervals. The clock overflows have to be
marked in the data stream read from the DPC module and be taken into regard by the SPCM
software. The raw data read from the DPC module are therefore difficult to interpret. Nevertheless, raw data can be stored by the SPCM software. Such data should, however, be used for
test purpose only.
To facilitate users to analyse time-tag data the SPCM software generates files in which all
these effects are already taken into regard. Please note that pre-processing does not cause any
56
SPCM Software
loss of photons or any loss in photon information. Pre-processed data are identified by
‘RAW = 0’. The description given below relates to pre-processed data.
Time per bin
TPB[23:0] defines the time unit in which all subsequent photon data are expressed. TPB is
given in femtoseonds. All times are expressed in multiples of TPB.
Records of Photons
The records following the descriptor are photon data. All detection events are recorded with
their channel number and a 54 bit integer time stamp. The time stamp reflects the absolute
time from the start of the measurement. The time unit is the TPB entry in the descriptor, see
Table 2.
To save disk space an event is normally stored with its detector channel number and the lower
24 bit of the time tag only. Only if the time in these 24 bits overflows the complete time is
written. The bits 30 and 31 are used to identify whether the record is a photon with a 24 bit
time or a record containing the higher bytes of the time of the previous photon. The meaning
of bit 30 and bit 31 are shown in Table 3.
Bit 31
Bit 30
Type
Description
0
0
Event
Low Part of Time [TPB] incl. channel number
0
1
TIME[53:24] Set Time to TIME[53:24]
1
0
Reserved
Reserved
1
1
Special
Descriptor, at start of file
Table 3: Bits identifying whether a record contains a 24 bit time or the higher bits for the complete 54 bit time
Event Record
The format of a photon record with 24-bit time is shown in Table 4.
Bit 31
Bit 30
Bit 29
Bit 28
Bit 27
Bit 26
Bit 25
Bit 24
0
0
GAP
Ch[4]
CH[3]
CH[2]
CH[1]
CH[0]
Bit 23
Bit 22
Bit 21
Bit 20
Bit 19
Bit 18
Bit 17
Bit 16
TIME[23]
TIME[22]
TIME[21]
TIME[20]
TIME[19]
TIME[18]
TIME[17]
TIME[16]
Bit 15
Bit 14
Bit 13
Bit 12
Bit 11
Bit 10
Bit 9
Bit 8
TIME[15]
TIME[14]
TIME[13]
TIME[12]
TIME[11]
TIME[10]
TIME[9]
TIME[8]
Bit 7
Bit 6
Bit 5
Bit 4
Bit 3
Bit 2
Bit 1
Bit 0
TIME[7]
TIME[6]
TIME[5]
TIME[4]
TIME[3]
TIME[2]
TIME[1]
TIME[0]
Table 4: Photon record (24 bit time)
CH[4:0]
Channel number of the DPC in which the photon was recorded. The LVTTL
have channel number from 1 to 16, the CFD channels have the channel numbers
1 and 2 for TDC 1 and 11 and 12 for TDC 2, see Fig. 77, page 47.
TIME[23:0] Event Time [TPB], low part. Only if bit 30 and bit 31 are 0, i.e. the higher part of
the time has not changed.
GAP
The FIFO has overflown. Data of a number of photons were lost before this entry.
Format of Time-Tag Data Files
57
High-Time Record
A record with bit 30 = 1 and bit 31 = 0 indicates that the higher part of the time has changed,
and that a new high-part of the photon time has to be used starting from the previously recorded photon, see Table 5. In other words, both parts, TIME[53:24] of this and TIME[23:0]
of the previous record, have to be used as a time tag.
Bit 31
Bit 30
Bit 29
Bit 28
Bit 27
Bit 26
Bit 25
Bit 24
0
1
TIME[53]
TIME[52]
TIME[51]
TIME[50]
TIME[49]
TIME[48]
Bit 23
Bit 22
Bit 21
Bit 20
Bit 19
Bit 18
Bit 17
Bit 16
TIME[47]
TIME[46]
TIME[45]
TIME[44]
TIME[43]
TIME[42]
TIME[41]
TIME[40]
Bit 15
Bit 14
Bit 13
Bit 12
Bit 11
Bit 10
Bit 9
Bit 8
TIME[39]
TIME[38]
TIME[37]
TIME[36]
TIME[35]
TIME[34]
TIME[33]
TIME[32]
Bit 7
Bit 6
Bit 5
Bit 4
Bit 3
Bit 2
Bit 1
Bit 0
TIME[31]
TIME[30]
TIME[29]
TIME[28]
TIME[27]
TIME[26]
TIME[25]
TIME[24]
Table 5: Record defining a new high-part of the photon time
Specification
LVTTL Inputs
No. of channels
Input Voltage
Threshold
Min. Input Pulse Width
Min. Pulse Distance
Connectors
16
LVTTL
1.4 V
2 ns
5.5 ns
MCX, on board
CFD Inputs
No of channels
Threshold
Zero Cross Adjust
Connectors
4
- 20 mV to - 500 mV
- 100 mV to + 100 mV
SMA, front panel
Experiment Trigger Input
Input Voltage
Threshold
Data Acquisition, Correlation Mode
Method
Correlation of photons
Autocorrelation
Cross-correlation
Time increment
Dead Time
No of parallel channels
On-board FIFO Buffer size
Readout
Sustained readout rate (typ., depends on computer)
LVTTL
1.4 V
Time-tag recording, absolute photon times
Multi tau or linear tau algorithm, online or offline
all channels
any pairs of channels
164.61 ps
< 10 ns
16 LVTTL or 4 CFD channels
4⋅106 photons
continuous readout during measurement
7⋅106 photons
Data Acquisition, TCSPC Mode
Method
Correlation of photons
Start (photon) channels
Dead Time
Stop channel
Stop input rate
Stop frequency divider
Time channel width
On-board FIFO Buffer size
Readout
Sustained readout rate (typ., depends on computer)
Time-tag recording, reversed start-stop
Start-stop histogram, online or offline
3 CFD inputs
< 10 ns
1 CFD input
max 150 MHz
1-2-4
164.61 ps
4⋅106 photons
continuous readout during measurement
7⋅106 photons
Data Acquisition, Multiscaler Mode
Method
Correlation of photons
Start (reference) channel
Stop (photon) channels
Dead Time
Time channel width
On-board FIFO Buffer size
Readout
Sustained readout rate (typ., depends on computer)
Time-tag recording, direct start-multistop
Start-stop histogram, online or offline
1 CFD input or 1 LVTTL input
3 CFD inputs or 15 LVTTL inputs
< 10 ns
164.61 ps
4⋅106 photons
continuous readout during measurement
7⋅106 photons
Operation Environment
Computer System
Recommended configuration
Bus Connector
Power Consumption
Dimensions
Pentium PC
>1024 Mb RAM, >100 Gb HD
PCI
approx. 12 W from +5V
312 mm x 124 mm x 20 mm
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg,
New York, 2005
W. Becker, The bh TCSPC Handbook. 3rd. edition, Becker & Hickl GmbH (2006) Available on
www.becker-hickl.com
Becker & Hickl GmbH, DCC-100 detector control module, manual, www.becker-hickl.com
Becker & Hickl GmbH, SPCImage Data Analysis Software for Fluorescence Lifetime Imaging Microscopy, available on www.becker-hickl.com
Becker & Hickl GmbH, BDL-375SMC, BDL-405SMC, BDL-440SMC, BDL-475SMC Ultraviolet and
Blue Picosecond Diode Lasers, available on www.becker-hickl.com
Becker & Hickl GmbH, DCS-120 Confocal Scanning FLIM Systems, user handbook. www.beckerhickl.com
Becker & Hickl GmbH, Modular FLIM systems for Zeiss LSM 510 and LSM 710 laser scanning microscopes. User handbook. Available on www.becker-hickl.com
K.M. Berland, P.T.C. So, E. Gratton, Two-photon fluorescence correlation spectroscopy, Method and
application to the intracellular environment, Biophys. J. 68, 694-701 (1995)
Y. Chen, J.D. Müller, P.T.C. So, E. Gratton, The Photon Counting Histogram in Fluorescence Fluctuation
Spectroscopy, Biophys. J. 77, 553-567 (1999)
Y. Chen, J.D. Müller, Q.Q. Ruan, E. Gratton, Molecular brightness characterization of EGFP in vivo by
fluorescence fluctuation spectroscopy, Biophys. J. 82, 133-144 (2002)
M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz and E. Gratton, Measuring Fast
Dynamics in Solutions and Cells with a Laser Scanning Microscope. Biophys. J. 89, 1317-1327 (2005)
S. Felekyan, R. Kühnemuth, V. Kudryavtsev, C. Sandhagen, W. Becker, C.A.M. Seidel, Full correlation
from picoseconds to seconds by time-resolved and time-correlated single photon detection, Rev. Sci. Instrum. 76, 083104 (2005)
S. Felekyan, Software package for multiparameter fluorescence spcetroscopy, full correlation amd multiparameter imaging. Available from www.mpc.uni-duesseldorf.de/seidel/software.htm
R. Hanbury-Brown, R.Q. Twiss, Nature 177, 27-29 (1956)
P. Kask, K. Palo, D.Ullmann, K. Gall, Fluorescence-intensity distribution analysis and its application in
bimolecular detection technology, PNAS 96, 13756-13761 (1999)
P. Kask, K. Palo, N. Fay, L. Brand, Ü. Mets, D. Ullmann, J. Jungmann, J. Pschorr, K. Gall, Twodimensional fluorescence intensity distribution analysis: theory and applications, Biophys. J. 78, 17031713 (2000)
P. Kask, C. Eggeling, K. Palo, Ü. Mets, M. Cole, K. Gall, Fluorescence intensity distribution analysis(FIDA) and related fluorescence fluctuation techniques: theory and practice, in R. Kraayenhof,
A.J.W.G. Visser, H.C. Gerritsen (eds.), Fluorescence spectroscopy inaging and probes, Springer Verlag
Berlin Heidelberg New York, 153-181 (2000)
Z. Kojro, A. Riede, M. Schubert, W. Grill, Systematic and statistical errors in correlation estimators obtained from various digital correlators, Rev. Sci. Instrum. 70, 4487-4496 (1999)
V. Kudryavtsev, S. Felekyan, A. K. Wozniak, M. König, C.Sandhagen, R.Kühnemuth, C. A. M. Seidel,
F. Oesterhelt, Monitoring dynamic systems with multiparameter fluorescence imaging. Anal Bioanal
Chem 387, 71–82 (2007)
D. Magde, E. Elson, W.W.W. Webb, Thermodynamic fluctuations ina reacting system - measurement by
fluorescence correlation spectroscopy, Phys. Rev. Lett. 29, 705-708 (1972)
J.D. Müller, Y. Chen, E. Gratton, Resolving Heterogeneity on the single molecular level with the photoncounting histogram, Biophys. J. 78, 474-586 (2000)
D.V. O’Connor, D. Phillips, Time-correlated single photon counting, Academic Press, London (1984)
K. Palo, L. Brand, C. Eggeling, S. Jäger, P. Kask, K. Gall, Fluorescence intensity and lifetime distribution
analysis: Toward higher accuracy in fluorescence fluctuation spectroscopy, Biophys. J. 83, 605-617
(2002)
K. Palo, Ü. Mets, S. Jäger, P. Kask, K. Gall, Fluorescence intensity multiple distribution analysis: concurrent determination of diffusion times and molecular brightness, Biophys. J. 79, 2858-2866 (2000)
M. Prummer, B. Sick, A. Renn, U.P. Wild, Multiparameter microscopy and spectroscopy for singlemolecule analysis, Anal. Chem. 76, 1633-1640 (2004)
H. Qian, E.L. Elson, Distribution of molecular aggregation by analysis of fluctuation moments, PNAS 87,
5479-5483 (1990)
R. Rigler, E.S. Elson (eds), Fluorescence Correlation Spectroscopy, Springer Verlag Berlin, Heidelberg,
New York (2001)
62
28.
29.
30.
31.
References
R. Rigler, J. Widengreen, Utrasensitive detection of single molecules by fluorescence correlation spectroscopy, Bioscience 3, 180-183 (1990)
P. Schwille, F.J. Meyer-Almes, R. Rigler, Dual-color fluorescence cross-correlation spectroscopy for
multicomponent diffusional analysis in solution, Biophys. J. 72, 1878-1886 (1997)
P. Schwille, U. Haupts, S. Maiti, W.W.W. Webb, Molecular Dynamics in Living Cells Observed by Fluorescence Correlation Spectroscopy with One- and Two-Photon Excitation, Biophys. J. 77, 2251-2265
(1999)
J. Widengren, V. Kudryavtsev, M. Antonik, S. Berger. M. Gerken, C. A. M. Seidel, Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal.Chem. 78,
2039-2050 (2006)
Index
2D FIDA 8
Absolute timing 2
Absolute timing mode 7
definition in the system parameters 45
Anti-bunching 31
combined with ps FCS 32
software setup 31
system parameters 31
A-PPI pulse inverter 22
Autocorrelation function 8
calculation from TDC data 9
normalisation 10
BDL-SMC picosecond diode laser 23, 25, 37
Beamsplitters, dichroic 27
Block Info 52
CFD inputs 20
Computer
installation of DPC 14
memory size 14
operating system 14
requirements to 14
Convert
FIFO files 54
Count rate
count rate display 44
Cross-correlation function 8
calculation from TDC data 9
normalisation 10
Cursors
during measurement 42
in the display window 42
Data analysis
send data to 43
Data and Setup File Formats 51, 52
Data file formats 55
DCS-120 confocal scanning FLIM system 35
Device driver 16
Device State 44
Dichroic beamsplitters 27
Display
cursors 42
display parameters 41
display windows in the main panel 39
of curve maximum 43
of first moment 43
of FWHM 43
of total counts 43
resizing of display window 42
runtime display 47
DPC-230
block diagram 2
buffer structure 3
CFD inputs 2, 20
device driver 16
FIFO 3
hardware Installation 15
installation 14
LVTTL inputs 2, 19
operation mode 45
Simple-Tau DPC system 1
software installation 14
specifications 59
Driver information 16
FCS 8, 26
autocorrelation 29
autocorrelation function 8
calculation from TDC data 9
cross-correlation 29
cross-correlation function 8
display of fit results 43
electronic setup 27
linear-tau algorithm 10, 48
main panel configuration 29
multi-tau algorithm 10, 48
normalisation 10
online calculation 47
online display 28
on-line display 47
online fit 28
optical setup 27
picosecond FCS 30
software setup 27
system parameters 27
trace parameters 29
two-photon 27
FIDA 8
FIFO
buffer structure 3
FIFO files 54
FIFO mode 13
Overflow 44
FIFO data, setup files 54
FIFO mode
FCS fit 28
File formats, data and setup 51, 52
File Info 51, 52
Files
conversion of FIFO files 54
FIFO 54
loading of 52
saving of 51
First moment
display of 43
FLIM 33
main panel configuration 35
microsecond FLIM 36
microsecond, laser control 37
microsecond, optical principle 36
microsecond, software setup 37
microsecond, system parameters 37
optical setup 33
scan clock pulses 33
software setup 34
software setup, µs FLIM 37
SPCImage data analysis 35
system parameter, µs FLIM 37
system parameters 34
Fluorescence correlation 8, 26
Fluorescence Decay Measurement 23
optical and electronical setup 23
software setup 23
system parameters 23
Fluorescence Lifetime Imaging 33
FWHM, display of 43
Hanbury-Brown-Twiss experiment 31
Hardware installation 15
Hardware Test 16
Initialisation Panel 16
64
Index
Inputs
CFD inputs 20
configuration of 49
for PMT modules with TTL output 19
for PMT pulses 21
for SPADs 19, 21
LVTTL inputs 19
marker pulses 20
synchronisation to light sources 20, 22
Installation 14
device driver 16
DLL library 15
hardware 15
software 14
Intensity traces 7
online calculation 48
Load 52
data files 52
FIFO files 54
file formats 52
load options 53
predefined setups 40, 53
setup files 52
Luminescence decay in the µs range 25
laser control 25
optical and electronical setup 25
software setup 26
system parameters 26
LVTTL inputs 19
Main Panel
count rate display 44
device state 44
Main panel of SPCM software
change between configurations 40
cursors 42
resizing panels 42
Main Panel of SPCM software
trace statistics 43
Marker pulses 20
Molecular brightness 8
Multichannel scaler mode 11
definition in the system parameters 46
PCH 7
online calculation 48
Photon counting histograms 7
Pinhole 27
PMTs
connecting to DPC 21
modules with TTL output 19
Predefined setups 40, 53
Rates 44
Relative timing 3, 11
Resizing Panels 42
Reversed start-stop 11
Runtime display 47
Save 51
data files 51
file formats 51
save setup data on exit 17
setup files 51
Simple-Tau DPC system 1
Simulation Mode 17
Software 39
Installation 14
simulation mode 17
start 16
start without module 17
SPADs
connecting to CFD inputs 22
connecting to DPC 19
pulse inverter 22
SPCImage data analysis 35
SPCM software 39
configuring the main panel 39
data file formats 55
display parameters 41
installation 14
Loading of files 52
online calculation 47
operation mode 45
predefined setups 40, 53
runtime display 47
saving of files 51
status information 44
system parameters 45
time-tag data file 46
Specifications of DPC-230 module 59
Synchronisation to Light Sources 20, 22
System parameters 45
CFD and SYNC parameters 49
input configuration 49
operation mode 45
runtime display 47
TDC parameters 50
TCSPC mode 3, 11
definition in the system parameters 45
FIFO 45
multi-hit capability 12
oscilloscope 45
reference frequency divider 4, 12
reversed start-stop 11
single 45
with absolute timing 13
TDC
definition of TDC parameters 50
principle 1
Time-tag mode
FCS fit 28
Time-tagged data 3, 9, 13, 55
file name 47
saving of time-tag data 27, 31, 46
Time-to-digital conversion 1
Timing
absolute timing 2, 7
frequency divider in reference channel 4
multichannel scaler mode 3, 11
reference pulses 4
relative timing 3, 11
TCSPC mode 4, 11
time-tagged data 3, 9, 13
Trace Statistics 43
Two-photon FCS 27
Waveform recording 3
Download PDF