Becker W. Advanced Time-Correlated Single Photon Counting Techniques

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X Contents

6.2 Characterisation of Detectors............................................... 222

6.2.1 Gain......................................................................... 222

6.2.2 Single-Electron Response ....................................... 222

6.2.3 Signal Transit Time ................................................ 224

6.2.4 Transit Time Spread................................................ 224

6.2.5 Pulse Amplitude Jitter............................................. 226

6.2.6 Cathode Efficiency.................................................. 229

6.2.7 Dark Count Rate ..................................................... 231

6.2.8 Afterpulsing Probability.......................................... 233

6.2.9 Prepulses ................................................................. 234

6.3 Measurement of PMT Parameters........................................ 234

6.3.1 Single Electron Response ....................................... 234

6.3.2 Transit Time Spread................................................ 236

6.3.3 Pulse Amplitude Distribution ................................. 237

6.3.4 Afterpulsing Probability.......................................... 239

6.3.5 Luminous Sensitivity and Quantum Efficiency ...... 241

6.4 Photon Counting Performance of Selected Detectors .......... 242

6.4.1 MCP-PMTs............................................................. 243

6.4.2 Cooled PMT Modules With GaAs Based

Cathodes.................................................................. 245

6.4.3 PMT Modules with Internal Discriminators ........... 247

6.4.4 Miniature PMTs in TO8 Housing......................... 248

6.4.5 Photosensor Modules.............................................. 249

6.4.6 Multianode PMTs ................................................... 252

6.4.7 Linear Focused PMTs ............................................. 254

6.4.8 Side-Window PMTs................................................ 256

6.4.9 Channel Photomultipliers........................................ 258

6.4.10 Single Photon Avalanche Photodiodes ................... 258

7 Practice of TCSPC Experiments ................................................. 263

7.1 Excitation Sources................................................................ 263

7.2 Optical Systems.................................................................... 268

7.2.1 Lenses ..................................................................... 269

7.2.2 Filters ...................................................................... 273

7.2.3 Beam Splitters......................................................... 278

7.2.4 Monochromators and Polychromators.................... 279

7.2.5 Optical Fibres.......................................................... 282

7.2.6 Reflections in Optical Systems ............................... 285

7.2.7 Baffles, Aperture Stops and Field Stops ................. 288

7.2.8 Detectors ................................................................. 289

7.2.9 Choosing the Detector............................................. 289

7.2.10 Quick Test of PMTs................................................ 292

Contents XI

7.2.11 Signal-Dependent Background ............................... 294

7.2.12 Instability in PMTs ................................................. 295

7.2.13 Timing Stability ...................................................... 296

7.2.14 PMT Voltage Dividers............................................ 299

7.2.15 Preamplifiers........................................................... 300

7.3 Detector Control and Overload Protection........................... 302

7.4 Generating the Synchronisation Signal ................................ 304

7.5 System Connections ............................................................. 307

7.5.1 Connector Systems.................................................. 307

7.5.2 Cables...................................................................... 308

7.5.3 Attenuators and Power Splitters.............................. 310

7.5.4 Shielding and Grounding ........................................ 311

7.6 Safety Considerations........................................................... 315

7.7 Setting the TCSPC System Parameters................................ 317

7.7.1 Optimisation of the CFD in the Detector

Channel ................................................................... 317

7.7.2 Reference CFD........................................................ 322

7.7.3 Adjusting the Delay in the Detector

and Reference Channel ............................................323

7.7.4 Choosing the TAC Parameters.................................326

7.8 Differential Nonlinearity .......................................................330

7.9 Counting Loss in TCSPC Systems........................................332

7.9.1 Classic Pile-Up Effect..............................................332

7.9.2 Counting Loss ..........................................................338

7.9.3 Dead-Time-Related Signal Distortion .....................343

7.10 Calibration of the Time Scale................................................345

8 Final Remarks................................................................................ 347

9 References ...................................................................................... 351

Index ............................................................................................... 389

List of Technical Terms and Symbols

ADC Analog-to-Digital Converter

AOM Acousto-Optical Modulator

APD Avalanche Photodiode. The diode achieves internal amplification

by using the avalanche effect for carrier multiplication.

laser Argon ion laser. A gas laser that emits at a number of wavelengths

in the blue-green region of the spectrum. When actively mode-

locked the Ar

laser delivers pulses of 80 to 125 MHz repetition

rate and 100 to 300 ps duration.

BNC A coaxial connector system, used for medium-bandwidth systems

in electronics.

Boxcar A signal-recording device based on sequential sampling of a re-

petitive, analog input signal. A large number of samples can be

averaged to recover a signal from the noise background.

CCD Charge-Coupled Device. The carriers are trapped under the gate

of a metal-oxide-semiconductor (MOS) structure, and shifted se-

quentially through the device. The technique is used for image

sensors.

CFD Constant-Fraction Discriminator. A CFD has a trigger delay

widely independent of the amplitude of the input pulses.

CFP Cyan fluorescent protein. CFP is a derivative of the green fluores-

cent protein, GFP. The fluorescent protein comes from a fluores-

cent jellyfish. The DNA of cells and whole organisms can be ma-

nipulated to produce these proteins.

Cj Symbol used for the junction capacitance of a semiconductor

diode

CLSM Confocal Laser Scanning Microscope. The sample is scanned by

a focused laser beam. The light from the excited spot is fed

through a pinhole in a plane optically conjugated with the focal

plane in the sample. The pinhole suppresses light from outside the

focal plane.

CMOS Complementary Metal Oxide Semiconductor technology, combi-

nation of N-channel and P-channel MOSFETs on the same chip.

The technique is the basis of all modern digital computing, mem-

ory, and logic circuits.

Counting Loss The loss of events (e.g. photons) in the dead time of the counter

after a previously detected photon.

Count rate Average number of events (e.g. photons) per unit of time. De-

tected count rate: Number of photon pulses per unit of time deliv-

XIV List of Technical Terms and Symbols

ered by the detector. Recorded count rate: Number of photons per

unit of time recorded by the photon counter.

CW Continuous Wave. Used for continuous electronic signals in the

radio-frequency range and continuous optical signals. In optics

the term is not quite uniformly used. Sometimes high-repetition

rate mode-locked lasers are called CW, although they emit a train

of pulses.

dB Logarithmic unit for relative voltage and power levels. For volt-

ages the decibel is dB = 20 log (V

), for powers dB = 10 log

)

Dead Time The time during which a counter or a detector is unable to accept a

new input event after the detection of a previous one.

Dichroic Selective reflection and transmission of light of different wave-

length. Dichroic mirrors are used to separate light signals of dif-

ferent wavelength.

DOT Diffuse Optical Tomography. The internal structure of highly

scattering objects is reconstructed by measuring the diffusely re-

flected and transmitted light in several directions.

DNL Differential Nonlinearity. The nonuniformity of the voltage in-

crements in an analog-to-digital converter, or the nonuniformity

of the time-channels of a photon counter

Dynode Amplification electrode in a photomultiplier tube (PMT). Elec-

trons are accelerated onto the dynode and multiplied by the emis-

sion of secondary electrons.

E Symbol used for „Efficiency“ in different contexts. The counting

efficiency describes the ratio of the number of photons needed by

an ideal optical recording technique to the number needed by a

real technique to obtain the same standard deviation of the result.

The FRET efficiency describes the coupling of donor-acceptor

pairs undergoing fluorescence-resonance energy transfer.

e Elementary charge (of the electron). 1,60210

-19

EB-CCD Electron-Bombarded CCD. A vacuum image sensor tube based

on a photocathode and a CCD sensor chip. The photoelectrons are

accelerated toward the CCD chip. A gain effect is obtained by

creating a large number of electron hole parts in the semiconduc-

tor for each incoming electron.

ECL Emitter-Coupled Logic. A logic family based on bipolar transistor

pairs connected via their emitters. ECL avoids saturation of the

transistors and has a signal delay widely independent of the tem-

perature. ECL is very fast but also power-consuming. It is used in

communication devices and fast timing electronics.

ECFP Enhanced cyan fluorescent protein. See CFP.

EMC Electromagnetic Compatibility

EYFP Enhanced yellow fluorescent protein. See YFP.

EROS Event Related Optical Signal. A fast change in the absorption and

scattering coefficients of the brain after stimulation.

List of Technical Terms and Symbols XV

F Symbol used for „Figure of Merit“. F describes the ratio of the

standard deviation of the fluorescence lifetime measured with an

ideal and a real recording technique.

FCS Fluorescence Correlation Spectroscopy. FCS uses the correlation

of fluctuations in the fluorescence intensity of a small number of

excited molecules.

FET Field-Effect Transistor. Junction-FETs exploit the change of the

cross-section of a conducting channel by the voltage-dependent

depletion region of a reverse-biased junction. MOSFETs modu-

late the carrier density in a conductive channel by the voltage ap-

plied to a metal-oxide-semiconductor structure.

FIDA Fluorescence Intensity Distribution Analysis. Uses the distribu-

tion of the fluorescence intensity of a small number of molecules

measured within consecutive time bins. See also PCH.

FILDA Fluorescence Intensity Distribution and Lifetime Analysis. Uses

the distribution of the fluorescence intensity and the fluorescence

lifetime of a small number of molecules measured within con-

secutive time bins.

FIFO First-In-First-Out. Principle used in data buffers. Successive data

words sent into the input port of a FIFO buffer are read in the

same order at the output port. FIFOs are used as temporary data

buffers in systems with fluctuating data rates.

FLIM Fluorescence Lifetime Imaging. The image is built up from the

fluorescence lifetime.

f number Ratio of the equivalent focal length of an optical system to the

diameter of the entrance pupil.

FPGA Field-Programmable Gate Array. Programmable logical device.

A large number of logic gates on a semiconductor chip is con-

nected by a programmed pattern to obtain a complex digital logic

system. FPGAs exist in one-time programmable and reprogram-

mable versions.

FRET Fluorescence Resonance Energy Transfer, or Förster Resonance

Energy Transfer. FRET is observed in systems with two fluoro-

phores when the emission band of the donor molecules overlaps

the absorption band of the acceptor molecules. If the donor is ex-

cited the energy is transferred directly into the acceptor and emit-

ted via the emission band of the acceptor. FRET occurs over dis-

tances of a few nm only.

FWHM Full Width at Half Maximum. Used as a definition of the electri-

cal and optical pulse width.

GaAs Gallium Arsenide. Semiconductor, used for high-frequency semi-

conductor components, for NIR photodiodes, and for photocath-

odes with high efficiency in the near infrared.

GaAsP Gallium Arsenide Phosphide. Material used for photocathodes

with high efficiency in the visible region.

XVI List of Technical Terms and Symbols

GFP Green fluorescent protein. GFP comes from a fluorescent jelly-

fish. The DNA of cells and whole organisms can be manipulated

to produce GFP.

GND Ground. Reference potential in electronic circuits.

HEK cell Human Embryonic Kidney cell. Cultured cells, frequently used

for biological experiments on the cell level.

HV High Voltage

I Symbol used for electrical current and light intensity

ICG Indocyanine Green. A contrast agent with absorption and fluores-

cence in the near infrared.

IR Infrared, spectral region from about 750 nm to 1 mm wavelength.

IRF Instrument Response Function. In a detection system the IRF is

the pulse shape obtained for an infinitely short input pulse. In a

fluorescence lifetime system the IRF is the pulse shape detected

for a sample with an infinitely short fluorescence lifetime.

Jitter Uncertainty in the time or amplitude of an optical or electrical

signal.

KDP Potassium Dihydrogen Phosphate. KDP crystals are used for

second-harmonic generation of laser radiation.

LED Light-Emitting Diode

LSM Laser Scanning Microscope

M1 First moment of a distribution. M1 of a photon distribution versus

time represents the average arrival time of the photons.

Macro Time Term used in time-correlated single photon counting. For each

individual photon, the time from the start of the experiment, the

Macro Time, and the time within the signal period, the Micro

Time, are stored.

MCA Multichannel Analyser. Records a histogram of the frequency of

the input pulses versus the amplitude.

MCP Multichannel Plate. A plate consisting of parallel, microscopi-

cally small channels oriented perpendicular to the surface. The

inner walls of the channels have a conductive coating. When a

voltage is applied between the opposite surfaces of the plate the

channels work as electron multipliers. MCPs are used in pho-

tomultiplier tubes and image intensifiers.

MCP-PMT Photomultiplier tube (PMT) based on electron multiplication in

multichannel plates.

MCS Multichannel Scaler. The device counts events, e.g. photons, into

successive channels of a fast memory.

MCX Connector system used in electronics.

Micro Time Term used in time-correlated single photon counting. For each

individual photon, the time from the start of the experiment, the

Macro Time, and the time within the signal period, the Micro

Time, are stored.

N Symbol used for the number of photons within a measurement

result, or within a defined time interval of a measurement result.

List of Technical Terms and Symbols XVII

NA Numerical Aperture. The sine of the vertex angle of the largest

cone of rays at the input or output of an optical system, multiplied

by the refractive index in which the cone is located. Defines the

light collecting power and the definition of an optical system.

NDD Nondescanned Detection. Detection principle used in laser-

scanning microscopes with multiphoton excitation. After passing

the objective lens, the light from the sample is diverted directly to

a large-area detector, without passing back through the scanner or

through a pinhole.

Nd:YAG Laser based on neodymium-doped yttrium-aluminium garnet.

The emission wavelength is 1,064 nm, the power can be up to sev-

eral tens of W. Mode-synchronisation delivers picosecond pulses

at a repetition rate of 50 to 100 MHz.

NIM Nuclear Instrumentation Module

NIR Near Infrared. Wavelength range from about 750 nm to 3 µm.

OPO Optical Parametric Oscillator. Uses nonlinear optical effects in a

crystal to split the pump beam into two coherent output beams of

tuneable wavelength. The sum of the reciprocal output wave-

lengths is equal to the reciprocal input wavelength.

PCB Printed Circuit Board

PCH Photon-Counting Histogram. Contains the distribution of the

fluorescence intensity of a small number of molecules measured

within consecutive time bins. The PCH is the basis of Fluores-

cence Intensity Distribution Analysis (FIDA).

PIN Diode Diode consisting of a positively doped (P) layer, an undoped (I)

layer, and a negatively doped (N) layer. Used for high-frequency

switches and attenuators, and for high-speed photodiodes.

PDT Photodynamic Therapy

Pile-Up In time-correlated single photon counting: Loss of a additional

photons detected after the first photon within one same signal pe-

riod. Pile-up causes distortion of the signal shape and loss in the

number of detected events. In high-energy particle detection: De-

tection of several particles within the response of a scintillator,

detector and subsequent amplifier. Pile-up causes distortion in the

measured energy distribution and loss in the number of detected

particles.

PLL Phase-Locked Loop. Circuit consisting of a phase comparator, a

loop filter, and a voltage-controlled oscillator. The phase com-

parator compares the phase of an input signal with the phase of

the oscillator signal. The oscillator is controlled as to maintain

zero phase between the oscillator and the input signal.

PMT Photomultiplier tube. Vacuum tube consisting of a photocathode

and a number of multiplication stages for the photoelectrons.

PMTs reach gains of 10

to 10

and are able to detect single pho-

tons.

QE Quantum Efficiency. The QE of a fluorophore is the ratio of the

number of emitted and absorbed photons. The QE of a photocath-

XVIII List of Technical Terms and Symbols

ode or a photodetector is the number of photoelectrons or the

number of detected photons divided by the number of incident

photons.

RET Resonance Energy Transfer. See FRET.

RG56, RG174 Standard types of coaxial cables with 50 : characteristic imped-

ance.

RF Radio Frequency

RMS Root mean square. Subsequent signal values are squared, the

mean of the squares is calculated, and the square root of that mean

is taken. Used to describe the uncertainty of a signal, the timing

accuracy of a detector or of an electronic system, or the deviation

of an optical surface from the ideal shape.

Routing Principle used in multidimensional time-correlated single photon

counting. Each photon is „routed“ into different memory blocks

according to a control signal read synchronously with its detec-

tion. Routing is used to record photons detected by several detec-

tors, to multiplex the measurement at different excitation wave-

lengths or sample positions, or to classify the photons according

to an externally measured parameter.

Router Device to operate several detectors at one time-correlated single

photon counting channel. The router exploits the fact that the de-

tection of several photon per signal period is unlikely. It combines

the photon pulses of all detectors into a common timing pulse line

and simultaneously generates a digital signal defining the detector

that detected the current photon.

Sequencer Programmable logic block in the recording electronics of multi-

dimensional time-correlated single photon counting. Used to re-

cord and accumulate sequences of recordings, or to acquire im-

ages in a scanning setup.

SER Single-Electron Response. Output pulse delivered by a detector

for a single photoelectron generated at its input. In most detectors

the SER is identical with the pulse shape for a single detected

photon.

SHG Second-Harmonic Generation. Generation of light of half the

wavelength of the incident laser light in a nonlinear crystal.

SLIM Spectral Lifetime Imaging. Combination of lifetime imaging

(FLIM) with simultaneous detection in several wavelength inter-

vals.

SMA A coaxial connector system, used for high-bandwidth connec-

tions in electronics.

SMB A coaxial connector system, used for medium-bandwidth connec-

tions in electronics

SNR Signal-to-Noise Ratio

SPAD Single photon Avalanche Photodiode. An avalanche photodiode

(APD) is operated above the breakdown voltage. A detected pho-

ton causes an avalanche breakdown with an easily detected cur-

rent pulse. SPAD operation requires an APD with uniform break-

List of Technical Terms and Symbols XIX

down over the full area and precautions to quench a triggered ava-

lanche and thus avoid destruction of the diode.

SPAPD Single photon Avalanche Photodiode, see SPAD.

TAC Time-to-Amplitude Converter. Converts the time between a start

and a stop pulse into a voltage. TACs can be built with a resolu-

tion down to a few picoseconds.

TCSPC Time-Correlated Single photon Counting. The technique is based

on the detection of single photons of the signal under investiga-

tion, measuring the detection times, and building up the distribu-

tion of the photon numbers versus the detection time.

TDC Time-to-Digital Converter. A circuit that converts the time of an

event, e.g. a detected photon, directly into its digital expression.

TDCs are normally based on the pulse delay in a chain of succes-

sive logic gates. The term is not quite uniformly used. Sometimes

modules based on analog time measurement principles with sub-

sequent analog-to-digital conversion are also called TDCs.

THG Third-Harmonic Generation. Generation of light of one third the

wavelength of the incident laser light in nonlinear crystals.

Ti:Sapphire Laser based on a titanium-doped sapphire crystal. The typical

power is a few watts, the wavelength tuneable from about 720 to

960 nm. The pulse width is in the range of 100 fs to a few ps, the

repetition rate between 78 and 92 MHz.

TOF Time-of-Flight. The term is used in experiments of high-energy

physics, mass spectroscopy, and diffuse optical tomography

(DOT). The TOF-distribution in DOT is the distribution of the

photons versus time after propagation through a turbid medium.

W Fluorescence lifetime spectroscopy: W is used for the fluorescence

lifetime or the lifetime components in multiexponential decay

functions. W

= natural lifetime in the (hypothetical) absence of all

nonradiative decay processes, W

= observed lifetime in the ab-

sence of external quenching processes, W

rot

= rotational depolarisa-

tion time.

Fluorescence correlation spectroscopy: W is used for the time in

the correlation function.

TTS Transit-Time Spread. In a photon counting detector, the transit

time for the individual photons varies. The TTS is the distribution

of the observed times of the output pulses for infinitely short input

light pulses.

UV Ultraviolet region of the optical spectrum, 1 nm to 400 nm.

YFP Yellow fluorescent protein. YFP is a derivative of the green fluo-

rescent protein, GFP. The fluorescent proteins come from a fluo-

rescent jellyfish. The DNA of cells and whole organisms can be

manipulated to produce these proteins.

1 Optical Signal Recording

100 years ago Einstein published his work „Über einen die Erzeugung und Ver-

wandlung des Lichtes betreffenden heuristischen Gesichtspunkt“ („On a Heuristic

Point of View about the Creation and Conversion of Light“) [157]. Since then it has

become a commonly known fact that light can be emitted and absorbed only in

discrete increments of energy. Emission and absorption phenomena can be ex-

plained only if light is considered as a stream of discrete particles, photons. Any

optical detection technique is subject to the same fundamental rules. Measurement

of light means absorption of photons in a detector. No matter how the detector

works and what the output signal of the detector is, the measurement represents a

discrete number of photons in a given time interval. For the majority of light signals

the individual photons are independent. That means, no detector and no optical

signal recording technique yields a standard deviation or a signal-to-noise ratio,

SNR, better than

NSNR (1.1)

for a number of photons, N, reaching the detector in a given time interval. For time-

resolved detection there is the obvious fact that, for a given light intensity, the num-

ber of photons is proportional to the detection time interval. Consequently, the sig-

nal-to-noise ratio decreases with decreasing time interval or increasing detection

bandwidth.

The effect is demonstrated in Fig. 1.1 for the output signal of a photomultiplier

tube. The figure shows the output signal of an XP2020 photomultiplier tube at a

light intensity corresponding to an average output current of –1 uA, –10 uA and

–100 uA (left to right). The bandwidth is 1 MHz, 10 MHz, and 100 MHz (top to

bottom).

A decrease is clearly visible in signal-to-noise ratio with increasing bandwidth.

Even more important, the output signal at high bandwidth is no longer a continuous

signal. Instead, it is a random sequence of pulses corresponding to the individual

photons detected. Even at 100 uA, the absolute maximum of the output current of

the XP2020 photomultiplier tube, the signal is not really continuous. This situation

is typical for optical detectors having a time resolution in the nanosecond and pico-

second range. At a gain high enough to distinguish single photons from the noise

floor and an average output current within the maximum load of the detector, the

output signal becomes a random sequence of pulses.

There are a number of techniques to recover the shape of the measured signal

from the detector output signal or to derive parameters of a system that is investi-

gated by a light signal. The efficiency of a signal recovering technique can be