4

Single Photon Counting

Yoshinori Mizuguchi

1. Introduction
Recently the demand for the measurement of very low-light levels has been
increasing m the fields of biology, chemistry, medicine, and many others; for
instance, analytical chemistry needs to employ fluorescence, luminescence and
Raman scattering light for the quantitative operation of substance.Biochemistry
calls for the measurement of very low-light level to observe gene expres-
sion (1,2) and the activity of living cell (3,4), etc. It also calls for radio-
immunoassay and fluorescent immunoassay. Medicme asksfor a positron com-
puterized tomography (CT) to research a brain physiologically and diagnose
cancers. In any case, the absolute levels of light to be detected are very low m
every field, so that light that changes with time at a very fast speed can be
captured.
The single photon-counting method counts photons of light individually,
which gives rise to higher stability and detectability, and also has the advan-
tage of the obtaining a superior signal-to-noise (S/N) ratio than the measure-
ment of direct current (DC) signals from detectors.
The principle of single photon counting is as follows. When incident light
enters the detector’s sensitive area, i.e., a photocathode, photoelectrons are
emitted from photocathode. These photoelectrons then travel through the elec-
tron multiplier, where electrons are multiplied by the process of secondary emis-
sion, and finally makes a pulse-shape signal as an output. If the intensity of
incident light to the detector is changed, the output signal becomes as shown in
Fig. 1. It is apparent from the figure that the pulse intervals become very narrow
in the regions of both high- and low-level light, where the output pulse overlap
with the analog pulse, i.e., DC signal is output (see Fig. 1A and B). At this light
level, the measurement of the DC-signal level is related to the intensity of the

From Methods m Molecular Biology, vol 108 Free Radrcal and Antroxrdant Protocols
Edlted by D Armstrong 0 Humana Press Inc , Totowa, NJ

37
38 Mizuguchi

Fig 1. Output pulses at difference light levels: (A) High, (B) Low, (C) Very Low.

incident light. However, as the intensity of the incident light becomes weak,
pulse-shape signal substances(dispersion) increase and finally become discrete
pulses (see Fig. 1C). At this light level, the measurement of the DC-signal level
is no longer related to the intensity of the Incident light, but the number of the
pulses per time is related the intensity of the incident light.
The method of processing output signals can be divided into analog mode
and digital mode. The measurement of these discrete pulses is called digital-
mode measurement, which is also called the photon-counting mode in general.
The process is called single-photon counting because the individual photon
signal can be detected. The measurement of DC-signal output at the high-light
level is called analog-mode measurement.
In the analog mode, the mean value containmg alternating current (AC)-
signal substances of Fig. 1A becomes a DC signal. On the other hand, the
photon-counting mode discriminates a pulse into binary signal, which is then
counted. The photon-counting mode therefore has many advantages and it is
considerably effective, espectally in the region of extremely low-light levels.
In order to perform the single-photon counting, a photomultiplier tube (PMT)
is commonly used as a detector. Recently, m order to obtain the image of the
photon signal from a sample, the two-dimensional photon-counting tube be-
came usable as a detector. This chapter is intended for those who want to famil-
iarize themselves with how to perform single-photon counting using PMT and
the two-dimensional photon-countmg tube. In this chapter, the operational prin-
ciples and the circuit configuration of these devices, as well as some notes, are
summarized. In addition, selection guides for these devices are discussed (5).
2. Materials
2.1. PMT
Among the photosensitive devices m use today, a PMT 1sa versatile device
that provides extremely high sensitivity and ultra-fast response. Figure 2 shows
a basic configuration of the photon-counting system. Because signal pulses
Single-Photon Counting 39

DISCRIMINATOR
VOLTAGE (COMPARATOR)
DIVIDER + PULSE SHAPER
PMT + CIRCUIT PREAMPLIFIER COUNTER
1:)CI‘,U
f-1 13fi fi
I
4

REFERENCE VOLTAGE
HIGH VOLTAGE

Fig. 2. Basrcconfiguration of photon-countmg system

from PMT are generally small, to amplify those pulses, a preamplifier is usu-
ally placed near the PMT for photon counting. Followed by the preamplifier, a
circuit drscriminator 1splaced to select out pulses whose height 1s higher, or
lower, than a certain height. Output pulses from the discriminator are then
shaped by a pulse shaper into pulses of constant width and herght. They are
finally counted by a counter.
2.1.1. PMT
A typical PMT consist of a photo-emissive cathode (photocathode) followed
by focusing electrodes, an electron multiplier, and an electron collector
(anode) in a vacuum tube, as shown in Fig. 3.
When light enters the photocathode, the photocathode emits photo-electrons
into the vacuum. These photo-electrons are then directed by the focusmg elec-
trode voltages towards the electron multiplier, where electrons are multiphed
by the process of secondary emission. The multiplied electrons are collected
by the anode as an output signal.
Because of secondary-emission multiplication, PMT provides extremely
high-sensitivity, exceptronally low noise among the photosensitrve devices
currently used to detect radiant energy in the ultraviolet (UV) and near infrared
(IR) region. PMT also features fast time response, low noise, and choice of
large photosensmve areas.
The section describes the prime features of PMT construction and basic
operating characteristics (6).
The photocathode of a PMT converts energy of incident light mto photo-
electrons. The conversion efficiency (photocathode sensitivity) varies with the
wavelength of the incident light. This relationship between photocathode
sensitivity and the wave-length is called the spectral-response characteristic.
40 Mizuguchi

FOCUSING
ELECTRODE
I

SECONDARY STEM PIN

LAST DYNODE
ELECTRON I

(l(r’ Pa)
/
DIREC TION OF LIGHT I
A E

FACEPLATE
\ STEM
ELECTORON
MULTIPLIER
(DYNODES)

\PHoToCATHODE

Fig 3 Cross-section of head-on type PMT

Figure 4 shows typical spectral response of btalkah PMT. The spectral-re-

sponse characteristics are determined on the long-wavelength side by the pho-
tocathode material and on the short-wavelength side by the window material
Actual data may be different from type to type.
As Fig. 4 shows, spectral response is usually expressed in terms of radiant
sensitivity or quantum efficiency as a function of wavelength. Radiant sensi-
tivity (S) is the photoelectrtc current from the photocathode, divided by the
incident radiant power at a given wavelength, expressed in amperes per watt
(A/W). Quantum efficiency (QE) is the number of photo-electrons emitted from
the photocathode divided by the number of the incident photons. It is custom-
ary to present QE m a percentage. QE and the radiant sensitivity have the fol-
lowing relationship at a given wavelength:
QE = (S x 1240)/(h) x 100 (%)
where S is the radiant sensmvtty m A/W at the given wavelength, and h is the
wavelength in nm (nano meters).
A low-noise electron multiplier, which amplifies electrons by a cascade sec-
ondary electron-emission process provtdes superior sensitivtty (high current
amphftcatton and high S/N) of PMTs. The electron multiplier consists of up to
about 16 stages of electrodes called dynodes (see Fig. 3). The photo-electron is
multiplied up to 106-107.
Though secondary electrons at first dynode come as a few discrete pulses
with respect to primary electron, they are regarded conloining to Poisson dis-
tribution, and the mean value of this dtstribution becomes a current amplifica-
 Single-Photon Counting 41

.
CATHODE
RADIANT
i SENSITIVITY
i

0.01’ I kl,
200 400 600 800

WAVELENGTH (r-m)

Fig. 4. Typical spectral response of head-on, bialkali photocathode.

tion (gain) o of PMT. This holds true for further multiplication processes from
the second dynode to the others. A set of the last electron group finally appears
as an output to the anode with respect to a single photon from the photocath-
ode. In a PMT that has n stage of dynodes, a smgle photoelectron from the
photocathode is multiplied by on as a mean, and the group of electrons makes
a pulse-shape output. Because of this, the gain of secondary electrons has var-
iance, i.e., statistical probability, and therefore the value of output pulse height
at the anode cannot be ahgned (see Fig. 5).
Furthermore, pulse heights at the anode are non-uniform owing to the non-
uniformrty of current amplifications caused by position difference of each dyn-
ode where electrons strike, and also owing to deviated electrons from the
42 Mizuguchi

PHOTOCAIHODE 1/ST DYNODE

I I I I

Ag. 5. Electron multrphcatron process by photon countmg mode

normal multiplication process. Heights of the pulse from the anode plotted m a
histogram are called a pulse-height distribution (PHD). Some examples are
illustrated in Fig. 6 which illustrates anode pulse having different height drs-
persed m time, integrated into a PHD. The abscissa of the figure represents
pulse heights, the number of electrons m a group, or the value of the pulse
voltage or current produced by that electron group. The PHD curve shows the
drstrrbutron of the value of voltage or current of the output of PMT.

2.1.2. Power Supply

The output of a PMT is extremely sensitive to the applied voltage by power
supply between the photocathode and the anode through the dynodes. Even
small variation m applied voltage greatly affect measurement accuracy. Thus,
a highly stable source of high voltage IS required. (see Fig. 7).
2.7.3. Preamplifer
Output pulses from a PMT m the single photon-counting mode are very low,
and it is therefore necessary to exercise great care m the design of preamplifier
and other circmts.

2.1.4. Counter
There are many types of counters, including the power supply, preamplifier,
pulse shaper, and drscrrmmator. There are many types of functtons from simple
counting to time-resolved photometry and computer control, etc A proper
counter can be selected for an application.

2.2. Two-Dimensional Photon-Counting Tubes

Recently, the demand for obtaining an image under very low-light levels has
been increasing, especially m the field of biology; for example in gene expression
Single-Photon Counting 43

f
PULSE HEIGHTS (CHARGES)

Fig. 6 PMT output and pulse height distribution.

(1,2), and the activity assay of living cell (34, etc. According to such demand,
several types of image sensors have been developed, “image intensifier” and
“cooled charge coupled device (CCD).” For the purpose of the single-photon
countmg, the image-intensifier type is more appropriate becauseof the extremely
low noise character compared to the CCD, and such an image intensifier is called
a two-dimensional photon-countmg tube. It is employed as one part of video
camera to get an image under the very low-light level (7).
Just like a PMT, a two-dimensional photon-countmg tube consist of a photo-
emissive cathode (photocathode) followed by focusing electrodes, and an elec-
tron multiplier m a vacuum tube, as shown in Fig. 8. The differences are the
44 Mizuguchi

10' 106

100 . 105

10-l 104

111-z 103
.- 200 300 500 700 1000 15oiI-

SUPPLY VOLTAGE (V)

Fig. 7 Example of the current amplification vs supply voltage.

phosphor screen and two or three stages of micro channel plate (MCP) employed
m two-dimensional photon-counhng tube instead of an electron corrector and
dynodes in PMT, respectively.
When incident light enters the photocathode, photo-electrons are emitted
from the photocathode. These photo-electrons are then directed by the focus-
ing electrode voltages towards the MCP, where electrons are multiplied to
about lo6 times by the process of secondary emission. The multlphed electrons
are bombarded to the phosphor screen and emit light as an output signal.
Because the relationship of the positIons is maintained from where photo-
electrons are first generated to where at last light signal is generated, the light
image that is proJected on the photocathode is obtained as an intensified image
Single-Photon Counting 45
PHOTON COUNTING IMAGER

EMISSION
4 MkP

‘PHOSPHOR When an electron IS lncldent

PHOl -0CATHODE MCP SCREEN on the inner walls of MCP, It IS
(ConversIon of (Multlpltcation of (Converslon of multtplled (or the number of
photon Into photoelectrons) photoelectron photoeletrons IS Increased) to
photoelectron) into light) about l,OOO,OOO electrons,
^^A Il.^.. ^*^ ^. .A....,

Fig. 8. Operatmg prmclple of two-dimensional photon-counting tube.

on the phosphor screen Therefore, this type of device 1s called an “image

intensifier.” The two-dimensional photon-counting tube is a type of the image
intensifier that has a high magnitude of multiplication with low noise.
As an important charactenstlcs, a two-dimensional photon-counting tube
also has a clear discrlmmator of the peak and valley in the PHD, the same as m
PMT; then it can be used for photon counting (see Fig. 9). An ordmary two-
dimensional photon-counting tube is coupled with a video camera through the
optics system, and the output image from the phosphor screen is read by a
video camera to get an image.
The video-camera system using two-dimensional photon-counting tube is
shown in Fig. 10.
3. Methods
3.1. Photon Counting on PMTs
3.1.1. Operation of Photon Counting
Because PMT output contams a variety of noise pulses in addition to the
signal pulse representing photo-electron as shown in Fig. 11, simply counting
pulses without some form of noise elimination will not result in accurate mea-
surement. The most effective approach to noise elimination is to investigate
the height of the output pulses.
A typical PHD for the output of PMTs is analyzed as shown in Fig. 12. In this
PHD, the lower-level dlscnmmation (LLD) is set at the valley through and the
upper-level discrlminatlon (ULD) at the foot, where the output pulses are very
46 Mizuguchi

ac

1 0’ I I 1
'0
+ PHOTON SPOTS BRIGHTNESS
(PULSE HEIGHT OF READ-OUT
PMT OUTPUT)
Fig 9 PHD of smgle photon spot (typical pulse-height distrtbutron of output spot
on output phospho screen).

2 Dnnensmnal Photon
Counting Tube
Image Processor

cl Video
Momtor

L Readout Video
Camera

Fig. 10 The block diagram of the vrdeo camera system

few. Most pulses lower than LLD are noise and pulses higher than ULD result
from cosmic rays, etc. Therefore, by counting pulses between the LLD and
ULD, accurate light measurements become possible. It is recommended that the
LLD be set at l/3 of the mean height of the pulses (Hm) and the ULD at three
times Hm. In most cases, however, the ULD settmg can be omitted.
Single-Photon Counting 47

COSMIC RAY PULSE

-----
DARK CURRENT SIGNAL PULSE

Fig. 11. Output pulse and discrimination level.

SIGNAL PULSE + NOISE PULSE

NOISE PULSE

LLD Hm ULD
PULSE HEIGHT

Fig. 12. Typical pulse-height distribution.

Considering the aforementioned, a clear definition of the peak and valley in

the PHD is a very significant characteristic for multiplier tubes for use in pho-
ton counting. Because of this, such types of PMTs are employed for use in
single-photon counting, having a high magnitude of multiplication and low
noise among the PMTs.
48 Mizuguchl

From the operational principle of photon countmg, the photon-counter system

and the multichannel pulse height analyzer systemhave been employed. Figure
13 shows the actual cn-cult configurations of each system and the pulse shapes
obtainable from circuns of each system.The photon-counter system of Fig. 13A
shows that output pulse from PMT are amplified by the preamplifier and further
amplified by the main amplifier, if necessary. Those amplified pulses are then
directed toward the discriminator, for which a comparator integrated circuit (IC)
is usually used. They are then compared with the pre-set reference voltage and
discnmmated into two groups of pulse. One group is lower and the other is higher
than the reference voltage. The lower pulses are finally eliminated by the LLD
and the higher pulses are eliminated by the ULD. The output of the comparator
takes place at a constant level (usually a transistor transistor logic [TTL] level
from O-5 V, and the output of a high-speed comparator takes place at an emttter-
coupled logic [ECL] level from -0.9 to -1.7). The counter connected followed by
the pulse shaper counts the number of those discriminated pulses only.
In contrast to the photon-counter system, in the multichannel pulse-height
analyzer system shown in Fig. 13B, output pulses from PMT are generally
integrated through a charge-sensitive-type preamplifier, amplified, and shaped
by the linear amplifier. These pulses are then drscrimmated by the discrimina-
tor. These discrimmated pulses are m turn converted from analog to digital.
They are finally accumulated by memory and displayed on the screen.
As shown m the figure, this system 1sable to output pulse-height informa-
tion and frequency (the number of counts) simultaneously.
The photon-counter system is used to measure the number of output pulse
from PMT corresponding to incident photons, whereas the multichannel pulse-
height discrimmator system is used to measure the height of each output pulse
and the number of output pulses simultaneously. The former system has a
superior feature of high-speed counting operation and so is used for general
purpose applications. Although the latter system has the disadvantage of
obtaining high counts, it is used for the application where a pulse-height
analysis is required; for example, radiation scmtillation counting and multi-
photon analysis
3.1.2. Setting the Discrimination Level
In the photon-counting mode, it is important to know where to set the dis-
crimination level
To optimize the S/N and the gam change rate, set a discrrminatron level near
the valley of a PHD curve or somewhere on a plateau of curve. Setting a dis-
crimmation level at as low a position as possible provides high counting effi-
ciency, but m reality it should be set near the valley of the PHD curve,
somewhere on a plateau of the plateau curve of the PMT. To eliminate noise
Single-Photon Counting 49

8’: n TTL LEVEL

PHOTON
r,i:::;:‘l_;r,
22 PMT CLR
DISCRIMINATOR PULSE SHAPER
(MAIN AMPLIFIER) (COMPARAToR) COUNTER
PREAMPLIFIER
LINEAR AMPLlf zIFR
._.. DISCRIMINATOR AID CONV ‘ERTER MEMORY, DISPLAY
PHOTON
52 1 [ ULD I
1 f 1
LLD I
1 1
I3 (CHARGE
SENSITIVE
AMP)

t-t
I ’* ULD
INTEGRATION
I :

*. -A- .. LLD

Fig. 13. Systems of photon counting: (A) Photon counter system, (B) multichannel
pulse-height analyzer system.

against signal, set the discrimination level at the middle point between the peak
and the valley of a PHD curve.
It is not easy to define a single position dlscnmination level, so it is neces-
sary to set a discrimination level according to the weight of the aforementioned
factors (see Notes l-4).
3.1.3. Noise
A very small amount of noise pulses exist in a PMT, even when it is kept in
complete darkness. This becomes an important factor in determinmg the lower
detection limit of a PMT. Noises depend upon the supply voltage. Thus, it 1s
suggested that the supply voltage should be as low as possible, if the amphflca-
tlon of the circuits permits. The origin of noise are as follows:
1. Emission of thermal electrons from photocathode and dynodes
2 Glass scmtlllatlons from the glass bulb of a PMT.
3. Leakage current owing to insulation failures of the socket and stem of a PMT
4. Noise by strong electric field da PMT IS operated at a voltage above the maxl-
mum-rated voltage
50 Mizuguchi

-60 -40 -20 0 20 40

AMBIENT TEMPERATURE (“C)

Fig. 14 Temperature characteristics of noise pulse

5 As an example of external noise, Fig. 14 shows the temperature characterlstlcs of

noise pulse of various photocathodes The temperature characteristic of PMTs
vary with respect to photocathode size and sensmvity, especially red-hght sense-
tivlty, etc

3.2. Photon Counting on Two-Dimensional Photon Counting

Tubes
In order to perform photon counting, the same process as with a PMT is
employed. The process is shown in Fig. 15 The incident photons are multi-
plied in the tube and turned into bright spots on the phosphor screen (Fig.
15A). The intensity of the video signal containing the bright spots are con-
verted to the digital signal and discriminated by a proper discrimination level
(Fig. 15B). At this time, the spots higher than the dlscrlminatlon level have the
Single-Photon Counting 51

0
0 0
0
A 0 0

:[
0 0 0
B Brightness
t

Fig. 15. The process m the video camera system. (A) Slgnal from video camera. (B)
Discrimination of signal. (C) The result of discrimmatlon.

value of 1, whereas the spots lower than the discrimmation level have the value
of 0 (Fig. 15C). Only the positions of the spots that have a value of 1 are stored
in the image memory.
3.3. Examples of the Applications
Photomultiplier tubes are widely used as photon-counting detectors; for
example, luminometers, spectrometers, and radioactivity detectors (such as
scintillation counter and posrtron CT, etc.).
According to the progress of the image-processing method, two-dimensional
photon-countmg tubes become useful m biomedical field; for example, gene
expression (1,2), the activity assay of living cell (3,4), and the use of a two-
dimensional luminometer to analyze samples srmultaneously
52 Mizuguchi
Zl
I I
IISSION-MODE +I - BOROSILICATE GLASS
--- UV,;GLASS 1 1
---- SYNTHETIC SILICA
TRANSMISSION-N
MULTIALKALI

ANSMISSION-MODE
QED MULTIALKALI

h)

10-Z
loo 300 500 700 900 1,100
WAVELENGTH (nm)
Rg 16. Spectral responsecharacteristics of typtcal photocathode.

4. Notes
1. Selectron guide: Because there are many types of detectors, tt is important to
select a proper detector for mdtvrdual application, based on materials such as
data sheetssupplied from the manufacturers. The basic characteristics to be con-
sidered are the spectral response,the noise charactertsttcs, the magnitude of the
multrphcatron of the signal, etc. The spectral-responsecharactenstrcsof typrcal
photocathodes are shown m Fig. 16
2 PHD and plateau characteristics The S/N ratio 1sone of the most important fac-
tors. Here, the S/N ISdefined asthe ratio of the mean value of signal counts to the
dispersionsof signal and noise counts. The S/N curve shown m Fig. 17 IS the
result of how the S/N changes when the supply voltage 1schanged Just as the
plateau charactertsttcs are obtained From a S/N point of view, tt 1sunderstand-
able that the supply voltage to the electron multrpher should be set between the
voltage (VO), which IS the starting point of the plateau region, and the maximum
rating of power supply voltage
Smgle-Photon Countmg

ETTING RAGE OF

SUPPLY VOLTAGE NV)

Fig. 17 An example of S/N curve of PMT.

Optimizmg the S/N* Fig. 18 shows the signal, the noise counts (integrated value),
and the characteristics of the ratio obtained when the discrrmmation level was
changed. This tell us that by setting the discrrmmation level L2 at about 200ch
(which corresponds to near the peak channel), it is possible to optimize
S/N ratio. In contrast, the analog mode does not have the capability of pulse-
height dtscrimmatton and therefore cannot take advantage of such a feature
Linearity of Count Rate. By nature, the photon-counting mode generally used in
the region of the very low-light level, and it has an excellent lmearity when the
count rate is low. However, the count rates becomes nonlmear when the photon-
counting mode is performed m the region under the large amounts of mcrdent light
A PMT itself has a wide band width, from 30 up to 300 MHz (which varies
depending upon the type of PMT used). This means that the maximum count rate
in the photon-countmg mode is determined mamly by the time resolution of an
electric crrcuit connected to the PMT
Because the time-interval distribution of photons that reach the PMT is a Poisson
distrrbution, the probability of counts (PO) by pulse overlaps, with respect to ran-
dom events (which corresponds to the normal photon countmg), can be obtained
using the followmg formula

PO = 1 - exp(-ZW)

where T 1sresolvmg time and R IS average count rate. Figure 19 shows the com-
parison between the measured value and the result of the formula above. It can be
seen that they match very well
54 Mizuguchi

PULSE HEIGHT (ch)

Ag 18 Opttmtzatron of S/N ratio

COUNT RATE (cps)

Fig. 19 Linearity of count rate

Thus, when the count rate 1shigh, tt 1snecessary to reduce them to below the
maximum count rate, consrdermgthe resolutton ttme of the ctrcmt parameter
Srmrlarly to PMT, a two-dtmensronal photon-countmg tube has nonlmear
characterrstrcswhen the count rate IShtgh Moreover when incident photons are
Single-Photon Counting 55

spatially concentrated at a region, a two-dimensional photon-countmg tube has

nonlinear characteristics, because the output photon spots overlap.

Acknowledgments
I thank Dr. Tadahisa Hiramttsu for reviewing this chapter. I also thank Mr.
Kiyoshi Kamlya and Mr. Toshihiro Kume for insight and helpful advice

References
1. Inoue, S (1992) Imagmg of luciferase secretion from transformed Chmese ham-
ster ovary cells. Proc. Natl. Acad. Scl. USA 85,9584-9587
2. Tamiya, E. (1990) Spatial imagmg of luciferase gene expression m transgemc
fish. Nucleic Acids Res 18(4), 1072.
3. Masuko, M. (1991) Rapid detection and counting of single bacteria m a wide field
using a photon countmg TV camera FEMS Microblol Lett. 83,23 l-238
4. Suematsu, M. (1987) Real-time visualtzatton of oxyradtcal burst from single neu-
trophil by using ultra-sensttive video intensifier microscopy Biochem Bzophys.
Res. Comm. 149(3), 1106-l 110.
5 How To Pe$orm Photon Counting Using Photomultiplier Tubes (1993) Hamamatsu
Photonics K K , 325-6, Sunayama-cho, Hamamatsu City, 430, Japan
6 Photomultiplier Tubes (1995) Hamamatsu Photomcs K K., 325-6, Sunayama-
cho, Hamamatsu City, 430, Japan.
7. Image Intensifiers (1992) Hamamatsu Photomcs K.K , 325-6, Sunayama-cho,
Hamamatsu City, 430, Japan