- Email: [email protected]
Development of an ultra-high sensitive photon counting system and its application to biomedical measurements
Optics and hers
in Engineering 3
(1982) 125-130
DEVELOPMENT OF AN ULTRA-HIGH SENSITIVE PHOTON COUNTING SYSTEM AND ITS APPLICATION TO BIOMEDICAL MEASUREMENTS H. INABA, A. YAMAGISHI,* C. TAKW Research
Institute
of Electrical Communication,
B. Department
of Medicine,
Tohoku
Tohoku Japan
YODA,
Y.
University,
Katahira
2-l-1,
Sendai
980,
GOTO
University, School of Medicine,
Seiryo-cho
l-l,
Sendai 980, Japan
T. MIYAZAWA, T. KANEDA Department
of Food Chemistry, Faculty of Agriculture, Tohoku Uniuersity, Amamiya-machi Sendai 980, Japan
l-1,
and A. SAEKI Tohoku Electronic Industrial Co. Ltd, Mukaiyama (Received:
20 January
2-36-4, Sendai 980, Japan
1982)
ABSTRACT
The sensitivity of the photon counting method for the detection of extra-weak optical intensity was improved on the introduction of the enhanced single photoelectron counting technique incorporated with a selected photomultiplier operated under optimum conditions. With this ultra-high sensitive photon counting system it has been possible to measure the difference between the luminescence of the blood of healthy and diseased human subjects (both have extremely weak values). With the development of optical electronics and a variety of its applications, the need to measure low intensities of light (from various types of faint source with low level photon emission) has become paramount. With this aim, the photon counting technique’ has been developed and is now customarily used to detect very weak light intensities (less than 10P’* to lo-” W) in the visible and *Present address: Toyonaka,
Osaka
High Magnetic 560, Japan.
Field
Laboratory,
Faculty
of
Science,
Osaka
University,
125 Optics and Lasers in Engineering Publishers Ltd, England, 1982 Printed in Northern Ireland
0143-8166/82/0003-0125/$02.75_0
Applied
Science
126
H. INABA
et al.
near infrared regions in conjunction with the high-sensitive photomultiplier. As a fascinating application of this method, we have measured extremely weak from various biological and chembioluminescence2 and chemiluminescence3-8 ical systems, such as living tissues like tumours and organs, and enzymatic reactions, and also analysed their extremely weak spectra using a specially However, the luminescence from blood designed spectral analyser system.‘.” and small pieces of tissue, for instance, is generally so weak that it is quite difficult to measure the intensity quantitatively with good reproducibility. This paper describes further improvement in the detection sensitivity of the photon counting system, particularly designed for biomedical and clinical applications, utilising the ‘enhanced single photoelectron counting method’, optimising the operational conditions and using carefully selected photomultipliers. By virtue of these improvements, we succeeded for the first time in detecting the extremely weak emission from human blood, the level of which should be useful for the discrimination of clinically diseased subjects from healthy ones in a sufficiently short time. In general, the optimum operational conditions of the photon counting method are achieved by maximising the figure of merit Mf defined by Mf = rl(A)F(u,)A,
(1)
where n(h) is the quantum efficiency of the photocathode, and A, is the effective photocathode area which is determined by the acceptance angle, the distance between the measured sample and the photocathode, and the actual photocathode area. F(u,J is expressed by
F(G) = Ds(21d)T1'2/[(nN)DN(2)d)11R where nd is a selected pulse-height discriminator voltage, (nN) is the average count rate of noise pulse and T is the measuring time. Ds(vJ and DN(ud) are the probabilities of occurrence of counting pulses, above the discriminator voltage u,+ caused by single photoelectrons and by noise current electrons, respectively. From eqns (1) and (2), it can be seen that it is essential to select a photomultiplier which possesses as large a value of D,(u,)/JD,(v,) as is possible, as well as a large photocathode area with low noise count rate, to give a high value of A,J&.J. The maximum value of D,(v,)/m is expected for a photomultiplier which has distinctly different pulse-height distributions for photoelectron and noise electron pulses. Although there exist photomultipliers designed for photon counting which have low noise count rate, the effective photocathode area is usually small (S 1 cm’) in order to achieve the desired count rate. We selected the specifically low noise count rate photomultiplier (typically about 60 counts/s at room temperature), from HTV
AN ULTRA-HIGH
SENSITIVE
PHOTON
COUNTING
SYSTEM
127
(Hamamatsu TV) R-878 photomultipliers, which employs a bialkali photocathode of 5 cm diameter with spectral response from nearly 300 nm to 660 nm. Its pulse-height distribution for photoelectron pulses, exhibiting a large bump corresponding to the single photoelectron event, is far from that of noise electron pulses which show almost an exponential curve. In order to achieve a higher value for the figure of merit with a given photomultiplier, it could be useful to emphasise the difference between the pulseheight distributions of photoelectron pulses and those of noise electron pulses by multiplying an appropriate pulse-height dependent function, G(v), by the pulse-height distribution function P(u). Usually G(v) should be an increasing function with pulse-height voltage TV.This newly proposed method called the ‘enhanced single photoelectron counting (ESPC) method’ is, for example, realised by inserting differentiating and square-law circuits between the photomultiplier and the pulse-height discriminator. After passing through these circuits, each electron pulse breaks into two pulses the height of which depends on the incident pulse-height. One can expect this to provide a larger value of D&u,)/-, which is different from its original value, depending on the P(u) values and the electron pulse shape. Based on this novel ESPC technique, we obtained, experimentally, a signal:noise ratio a little higher than that for the conventional photon counting method. A schematic diagram of the ultra-high sensitive photon counting system, thus realised for biomedical and clinical applications, is shown in Fig. 1. To attain a larger effective photocathode area, A,, for the photomultiplier, the distance between the photocathode and the sample cell is minimised (to about 8 cm), such adjustments are, however, limited due to the space required for a chopping wheel, coloured glass (or interference) filters and two glass windows. In addition, the ageing period of about a week for the photomultiplier was sufficient to achieve the low noise count rate, which decreased to 10 counts/s at -20°C and was very stable against variations in the ambient temperature. Furthermore, by shielding the photomultiplier and electronics completely from surrounding noise sources, we could realise a 10 to 20 times higher value for the figure of merit, M,, for the new photon counting system compared to that for the previous photon counting apparatus.” The first measurement performed, to our knowledge, was that of the extremely weak chemiluminescence intensity from human bloods using the ESPC system shown in Fig. 1. Two 1 ml heparinised blood samples of normal and diseased subjects were placed in a stainless steel sample cell of about 5 cm diameter. The single photoelectron pulses were counted for a period of 300 s with the sample at 37°C by the synchronous ESPC method incorporating the chopping wheel and bi-directional counter. Figure 2 depicts the results of a series of measurements. Blood samples of healthy subjects gave a low count rate, sometimes close to zero; the mean value was 470 counts/300 s. On the
128
et Cd.
H. INABA
DELAY
DIFFERENTIATING AND SQUARELAW CIRCUITS
1 PRE-AMPLIFIER
I
L__---_-_-_----_-----
Fig. 1.
Schematic
diagram
LINE
GATE CONTROL
1
I
I
----
of the ultra-high sensitive photon counting for biomedical and clinical applications.
-l
system
specially
designed
other hand, the samples from patients with diabetes mellitus showed 3-4 times higher emission levels than those of the control samples from healthy people (average count rate: 1640 counts/300 s). The blood glucose levels of diabetic subjects bore no direct relationship to the emission intensity of their blood samples. Blood samples from patients with carcinomas also showed a 3-4 times higher count rate (average count rate: 1710 counts/300s). Patients with carcinomas in the hepato-biliary system tended to show higher emission levels from their blood than those patients with carcinomas in gastro-intestinal
AN ULTRA-HIGH
SENSITIVE
PHOTON
COUNTING SYSTEM
129
tracts. Hyperlipidemic subjects generally exhibited a higher count rate from their blood. Though the exact identity and clinical significance of the extremely weak light-emitting substances in human blood samples are not yet known, they are most likely to be due to increased amounts of activated oxygen and radicals,ll*l’ such as singlet oxygen or related substances such as those responsible for lipid peroxidation, and/or the decreased degree of anti-oxidative activity in the blood. Measurements made with the ultra-high sensitive photon counting system presented in this paper are being continued extensively to find further applications in the fields of medicine and bio-science. The detailed results will be published elsewhere in the future.
:.......::..:: .. ,. .. ,. ..
NORMAL
CANCER
:. .:. :::.:::.;: ::>:.: ::: - -
DIABETES MELLITUS
::::....:.:. :.,;.:;.::,::;::> : ..;::: .A...,
HYPERLIPIDEMIA
Fig. 2. Measured results of extremely weak luminescence intensity from blood samples taken from healthy people (number of samples, n = 13) and from patients with cancer (n = 12), diabetes mellitus (n = 13) and hyperlipidemia (n = 7).
130
H. INABA et cd. ACKNOWLEDGEMENTS
This work was supported in part by a Scientific Research Grant-In-Aid for Co-operative Research (No. 539002) in 1980 from the Ministry of Education, Science and Culture of Japan.
REFERENCES
1. 2.
3.
4.
5. 6.
7.
8.
9. 10.
11. 12.
E. G. MORTON, Photon counting, Appl. Opt., 7 (1968) pp. l-10. Y. SHIMIZU, H. INABA, K. KUMAKI, K. MIZLJNO. S. HATA and S. TOMIOKA. Measuring methods for ultra-low light intensity and their application to extra-weak spontaneous bioluminescence from living tissues, IEEE Trans. Inst. Meas., IM-22 (1973) pp. 153-7. M. NAKANO, T. NOGUCHI, K. SUGIOKA, H. FUKWAMA, M. SATO, Y. SHIMIZU, Y. TSUJI and H. INABA, Spectroscopic evidence for the generation of singlet oxygen in the reduced nicotinamide adenine dinucleotide phosphate-dependent microsomal lipid peroxidation system, J. Biolog. Chem., 250 (1975) pp. 2404-6. M. NAKANO, K. TAKAYAMA, Y. SHIMIZU, Y. TSUJI, H. INABA and T. MIGITA, Spectroscopic evidence for the generation of singlet oxygen in self-reaction of set-peroxy radicals, J. Amer. Chem. Sot., 98 (1976) pp. 1974-5. Y. USHIJIMA, M. NAKANO, Y. TSUJI and H. INABA, Excitation of indole analogs by phagocytosing leukocytes, B&hem. Biophys. Rex Commun., 82 (1978) pp. 853-8. S. KOBAYASHI, K. SUGIOKA, M. NAKANO, C. TAKYU, A. YAMAGISHI and H. INABA, Excitation of indole acetate in myeloperoxidase-H,O, system: possible formation of indole acetate cation radical, Biochem. Biophys. Res. Comm., 93 (1980) pp. 967-73. W. ANDO, Y. KATE, S. KOBAYASHI, C. TAKYU, A. YAMAGISHI and H. INABA, Formation of sulfinyl oxide and singlet oxygen in the reaction of thianthrene cation radical and superoxide ion, J. Amer. Chem. Sot., 102 (1980) pp. 4526-8. T. YOSHIMOTO, S. YAMAMOTO, K. SUGIOKA, M. NAKANO, C. TAKYU, A. YAMAGISHI and H. INABA, Studies on the tryptophan-dependent light emission by prostaglandin hydroperoxidase reaction, J. Biolog. Chem., 255 (1980) pp. 10199-204. H. INABA, Y. SHIMIZU and Y. TSUJI, Measurement of very weak light signals and spectra, Japan. J. Appl. Phys., 14 (Suppl. 14-l) (1974) pp. 23-32. H. INABA, Y. SHIMIZU, Y. TSUJI and A. YAMAGISHI, Photon counting spectral analyzing system of extra-weak chemi- and bio-luminescence for biochemical applications, Photochem. Photobiol., 30 (1979) pp. 169-75. 0. HAYAISHI and K. ASADA (ed.), Biochemical and Medical Aspects of Active Oxygen, Tokyo, Japan Scientific Societies Press, 1977. H. H. WASSERMAN and R. W. MURRAY (eds.), Singlet Oxygen, New York, Academic Press, 1979.
in Engineering 3
(1982) 125-130
DEVELOPMENT OF AN ULTRA-HIGH SENSITIVE PHOTON COUNTING SYSTEM AND ITS APPLICATION TO BIOMEDICAL MEASUREMENTS H. INABA, A. YAMAGISHI,* C. TAKW Research
Institute
of Electrical Communication,
B. Department
of Medicine,
Tohoku
Tohoku Japan
YODA,
Y.
University,
Katahira
2-l-1,
Sendai
980,
GOTO
University, School of Medicine,
Seiryo-cho
l-l,
Sendai 980, Japan
T. MIYAZAWA, T. KANEDA Department
of Food Chemistry, Faculty of Agriculture, Tohoku Uniuersity, Amamiya-machi Sendai 980, Japan
l-1,
and A. SAEKI Tohoku Electronic Industrial Co. Ltd, Mukaiyama (Received:
20 January
2-36-4, Sendai 980, Japan
1982)
ABSTRACT
The sensitivity of the photon counting method for the detection of extra-weak optical intensity was improved on the introduction of the enhanced single photoelectron counting technique incorporated with a selected photomultiplier operated under optimum conditions. With this ultra-high sensitive photon counting system it has been possible to measure the difference between the luminescence of the blood of healthy and diseased human subjects (both have extremely weak values). With the development of optical electronics and a variety of its applications, the need to measure low intensities of light (from various types of faint source with low level photon emission) has become paramount. With this aim, the photon counting technique’ has been developed and is now customarily used to detect very weak light intensities (less than 10P’* to lo-” W) in the visible and *Present address: Toyonaka,
Osaka
High Magnetic 560, Japan.
Field
Laboratory,
Faculty
of
Science,
Osaka
University,
125 Optics and Lasers in Engineering Publishers Ltd, England, 1982 Printed in Northern Ireland
0143-8166/82/0003-0125/$02.75_0
Applied
Science
126
H. INABA
et al.
near infrared regions in conjunction with the high-sensitive photomultiplier. As a fascinating application of this method, we have measured extremely weak from various biological and chembioluminescence2 and chemiluminescence3-8 ical systems, such as living tissues like tumours and organs, and enzymatic reactions, and also analysed their extremely weak spectra using a specially However, the luminescence from blood designed spectral analyser system.‘.” and small pieces of tissue, for instance, is generally so weak that it is quite difficult to measure the intensity quantitatively with good reproducibility. This paper describes further improvement in the detection sensitivity of the photon counting system, particularly designed for biomedical and clinical applications, utilising the ‘enhanced single photoelectron counting method’, optimising the operational conditions and using carefully selected photomultipliers. By virtue of these improvements, we succeeded for the first time in detecting the extremely weak emission from human blood, the level of which should be useful for the discrimination of clinically diseased subjects from healthy ones in a sufficiently short time. In general, the optimum operational conditions of the photon counting method are achieved by maximising the figure of merit Mf defined by Mf = rl(A)F(u,)A,
(1)
where n(h) is the quantum efficiency of the photocathode, and A, is the effective photocathode area which is determined by the acceptance angle, the distance between the measured sample and the photocathode, and the actual photocathode area. F(u,J is expressed by
F(G) = Ds(21d)T1'2/[(nN)DN(2)d)11R where nd is a selected pulse-height discriminator voltage, (nN) is the average count rate of noise pulse and T is the measuring time. Ds(vJ and DN(ud) are the probabilities of occurrence of counting pulses, above the discriminator voltage u,+ caused by single photoelectrons and by noise current electrons, respectively. From eqns (1) and (2), it can be seen that it is essential to select a photomultiplier which possesses as large a value of D,(u,)/JD,(v,) as is possible, as well as a large photocathode area with low noise count rate, to give a high value of A,J&.J. The maximum value of D,(v,)/m is expected for a photomultiplier which has distinctly different pulse-height distributions for photoelectron and noise electron pulses. Although there exist photomultipliers designed for photon counting which have low noise count rate, the effective photocathode area is usually small (S 1 cm’) in order to achieve the desired count rate. We selected the specifically low noise count rate photomultiplier (typically about 60 counts/s at room temperature), from HTV
AN ULTRA-HIGH
SENSITIVE
PHOTON
COUNTING
SYSTEM
127
(Hamamatsu TV) R-878 photomultipliers, which employs a bialkali photocathode of 5 cm diameter with spectral response from nearly 300 nm to 660 nm. Its pulse-height distribution for photoelectron pulses, exhibiting a large bump corresponding to the single photoelectron event, is far from that of noise electron pulses which show almost an exponential curve. In order to achieve a higher value for the figure of merit with a given photomultiplier, it could be useful to emphasise the difference between the pulseheight distributions of photoelectron pulses and those of noise electron pulses by multiplying an appropriate pulse-height dependent function, G(v), by the pulse-height distribution function P(u). Usually G(v) should be an increasing function with pulse-height voltage TV.This newly proposed method called the ‘enhanced single photoelectron counting (ESPC) method’ is, for example, realised by inserting differentiating and square-law circuits between the photomultiplier and the pulse-height discriminator. After passing through these circuits, each electron pulse breaks into two pulses the height of which depends on the incident pulse-height. One can expect this to provide a larger value of D&u,)/-, which is different from its original value, depending on the P(u) values and the electron pulse shape. Based on this novel ESPC technique, we obtained, experimentally, a signal:noise ratio a little higher than that for the conventional photon counting method. A schematic diagram of the ultra-high sensitive photon counting system, thus realised for biomedical and clinical applications, is shown in Fig. 1. To attain a larger effective photocathode area, A,, for the photomultiplier, the distance between the photocathode and the sample cell is minimised (to about 8 cm), such adjustments are, however, limited due to the space required for a chopping wheel, coloured glass (or interference) filters and two glass windows. In addition, the ageing period of about a week for the photomultiplier was sufficient to achieve the low noise count rate, which decreased to 10 counts/s at -20°C and was very stable against variations in the ambient temperature. Furthermore, by shielding the photomultiplier and electronics completely from surrounding noise sources, we could realise a 10 to 20 times higher value for the figure of merit, M,, for the new photon counting system compared to that for the previous photon counting apparatus.” The first measurement performed, to our knowledge, was that of the extremely weak chemiluminescence intensity from human bloods using the ESPC system shown in Fig. 1. Two 1 ml heparinised blood samples of normal and diseased subjects were placed in a stainless steel sample cell of about 5 cm diameter. The single photoelectron pulses were counted for a period of 300 s with the sample at 37°C by the synchronous ESPC method incorporating the chopping wheel and bi-directional counter. Figure 2 depicts the results of a series of measurements. Blood samples of healthy subjects gave a low count rate, sometimes close to zero; the mean value was 470 counts/300 s. On the
128
et Cd.
H. INABA
DELAY
DIFFERENTIATING AND SQUARELAW CIRCUITS
1 PRE-AMPLIFIER
I
L__---_-_-_----_-----
Fig. 1.
Schematic
diagram
LINE
GATE CONTROL
1
I
I
----
of the ultra-high sensitive photon counting for biomedical and clinical applications.
-l
system
specially
designed
other hand, the samples from patients with diabetes mellitus showed 3-4 times higher emission levels than those of the control samples from healthy people (average count rate: 1640 counts/300 s). The blood glucose levels of diabetic subjects bore no direct relationship to the emission intensity of their blood samples. Blood samples from patients with carcinomas also showed a 3-4 times higher count rate (average count rate: 1710 counts/300s). Patients with carcinomas in the hepato-biliary system tended to show higher emission levels from their blood than those patients with carcinomas in gastro-intestinal
AN ULTRA-HIGH
SENSITIVE
PHOTON
COUNTING SYSTEM
129
tracts. Hyperlipidemic subjects generally exhibited a higher count rate from their blood. Though the exact identity and clinical significance of the extremely weak light-emitting substances in human blood samples are not yet known, they are most likely to be due to increased amounts of activated oxygen and radicals,ll*l’ such as singlet oxygen or related substances such as those responsible for lipid peroxidation, and/or the decreased degree of anti-oxidative activity in the blood. Measurements made with the ultra-high sensitive photon counting system presented in this paper are being continued extensively to find further applications in the fields of medicine and bio-science. The detailed results will be published elsewhere in the future.
:.......::..:: .. ,. .. ,. ..
NORMAL
CANCER
:. .:. :::.:::.;: ::>:.: ::: - -
DIABETES MELLITUS
::::....:.:. :.,;.:;.::,::;::> : ..;::: .A...,
HYPERLIPIDEMIA
Fig. 2. Measured results of extremely weak luminescence intensity from blood samples taken from healthy people (number of samples, n = 13) and from patients with cancer (n = 12), diabetes mellitus (n = 13) and hyperlipidemia (n = 7).
130
H. INABA et cd. ACKNOWLEDGEMENTS
This work was supported in part by a Scientific Research Grant-In-Aid for Co-operative Research (No. 539002) in 1980 from the Ministry of Education, Science and Culture of Japan.
REFERENCES
1. 2.
3.
4.
5. 6.
7.
8.
9. 10.
11. 12.
E. G. MORTON, Photon counting, Appl. Opt., 7 (1968) pp. l-10. Y. SHIMIZU, H. INABA, K. KUMAKI, K. MIZLJNO. S. HATA and S. TOMIOKA. Measuring methods for ultra-low light intensity and their application to extra-weak spontaneous bioluminescence from living tissues, IEEE Trans. Inst. Meas., IM-22 (1973) pp. 153-7. M. NAKANO, T. NOGUCHI, K. SUGIOKA, H. FUKWAMA, M. SATO, Y. SHIMIZU, Y. TSUJI and H. INABA, Spectroscopic evidence for the generation of singlet oxygen in the reduced nicotinamide adenine dinucleotide phosphate-dependent microsomal lipid peroxidation system, J. Biolog. Chem., 250 (1975) pp. 2404-6. M. NAKANO, K. TAKAYAMA, Y. SHIMIZU, Y. TSUJI, H. INABA and T. MIGITA, Spectroscopic evidence for the generation of singlet oxygen in self-reaction of set-peroxy radicals, J. Amer. Chem. Sot., 98 (1976) pp. 1974-5. Y. USHIJIMA, M. NAKANO, Y. TSUJI and H. INABA, Excitation of indole analogs by phagocytosing leukocytes, B&hem. Biophys. Rex Commun., 82 (1978) pp. 853-8. S. KOBAYASHI, K. SUGIOKA, M. NAKANO, C. TAKYU, A. YAMAGISHI and H. INABA, Excitation of indole acetate in myeloperoxidase-H,O, system: possible formation of indole acetate cation radical, Biochem. Biophys. Res. Comm., 93 (1980) pp. 967-73. W. ANDO, Y. KATE, S. KOBAYASHI, C. TAKYU, A. YAMAGISHI and H. INABA, Formation of sulfinyl oxide and singlet oxygen in the reaction of thianthrene cation radical and superoxide ion, J. Amer. Chem. Sot., 102 (1980) pp. 4526-8. T. YOSHIMOTO, S. YAMAMOTO, K. SUGIOKA, M. NAKANO, C. TAKYU, A. YAMAGISHI and H. INABA, Studies on the tryptophan-dependent light emission by prostaglandin hydroperoxidase reaction, J. Biolog. Chem., 255 (1980) pp. 10199-204. H. INABA, Y. SHIMIZU and Y. TSUJI, Measurement of very weak light signals and spectra, Japan. J. Appl. Phys., 14 (Suppl. 14-l) (1974) pp. 23-32. H. INABA, Y. SHIMIZU, Y. TSUJI and A. YAMAGISHI, Photon counting spectral analyzing system of extra-weak chemi- and bio-luminescence for biochemical applications, Photochem. Photobiol., 30 (1979) pp. 169-75. 0. HAYAISHI and K. ASADA (ed.), Biochemical and Medical Aspects of Active Oxygen, Tokyo, Japan Scientific Societies Press, 1977. H. H. WASSERMAN and R. W. MURRAY (eds.), Singlet Oxygen, New York, Academic Press, 1979.