Normalization technique for photon-counting luminometer

• US 5,321,261 A
• Filed: 09/10/1992
• Issued: 06/14/1994
• Est. Priority Date: 09/10/1992
• Status: Expired due to Term

• Current Assignee: Packard Instrument Co., Inc. (Revvity, Inc.)
• Original Assignee: Packard Instrument Co., Inc. (Revvity, Inc.)
• Inventors: Valenta, Robert J.
• Primary Examiner(s): FIELDS, CAROLYN
• Application Number:
  US07/943,398
• Time in Patent Office: 642 Days
• Field of Search: 250/252.1 R, 250/328, 250/394, 250/366, 250/369, 250/363.09, 250/361 C
• US Class Current: 250/252.1
• CPC Class Codes: G01J 1/08 Arrangements of light sources specially adapted for photometry standard sources, also using luminescent or radioactive material
• CPC Class Codes: G01J 1/18 using comparison with a reference electric value
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01T 1/40 Stabilisation of spectrometers
• CPC Class Codes: G01T 7/00 Details of radiation-measuring instruments

Abstract

A normalization system for multi-photodetector single photon counting luminescent measuring systems and a standard for use in the same. The method comprises calculating one or more counting ratios for each photodetector in the single photon counting system and multiplying counts of unknown samples by the inverse of the appropriate counting ratio. A counting ratio for a particular photodetector is calculated by dividing the count of a standard measured by the photodetector by the count of the same standard measured by a reference photodetector. The particular standard developed employs calcium tungstate:lead as a scintillator and tritium or carbon-14 thymidine as a means of exciting the scintillator. This standard has the advantages of providing a good model of typical luminescence chemistry and producing an emission pattern which is susceptible to single photon counting.

Claims

1. A method for normalizing two or more photodetectors in a single-photon counting system for measuring the luminescence of multiple samples, said photodetectors comprising a reference detector and one or more other photodetectors, said method comprising:

successively counting the photon emissions of a standard by two or more photodetectors, at least one of said photodetectors being the reference detector;

computing a counting ratio for each of said one or more other photodetectors, said counting ratio being defined as the ratio of counts from the corresponding one or more other photodetectors to the counts from the reference detector; and

in subsequent use of the photodetection system, multiplying the counts of a luminescent sample measured by a particular photodetector by the inverse of said counting ratio computed for that particular photodetector.

2. The method of claim 1 wherein the photodetectors are photomultiplier tubes.

3. The method of claim 2 wherein the sample is chemiluminescent.

4. The method of claim 1 wherein the standard comprises:

a scintillator with a peak wavelength approximately the same as that of said luminescent sample and a long decay constant,

one or more radioactive materials,

and a containment means.

5. The method of claim 4 wherein said scintillator has a decay constant greater than about 10 μ

s.

6. The method of claim 5 wherein said scintillator comprises calcium tungstate:

lead.

7. The method of claim 4 wherein the standard additionally comprises a means for maintaining said scintillator and said one or more radioactive materials in said containment means.

8. The method of claim 7 wherein said means for maintaining said scintillator and said one or more radioactive materials in said containment means includes a binder material.

9. The method of claim 8 wherein said binder material is carboxy methyl cellulose.

10. The method of claim 7 wherein said means for maintaining said scintillator and said one or more radioactive materials in said containment means includes a potting material.

11. The method of claim 7 wherein said means for maintaining said scintillator and said one or more radioactive materials in said containment means includes a binder material and a potting material.

12. The method of claim 11 wherein said scintillator comprises calcium tungstate:

lead and said one or more radioactive materials is either tritium or carbon-14 thymidine.

13. The method of claim 4 wherein said one or more radioactive materials is either tritium or carbon-14 thymidine.

14. A method for normalizing two or more photodetectors in a single-photon counting system for measuring the luminescence of multiple samples, said method comprising:

successively counting the photons emitted by a standard by each of said photodetectors;

computing a ratio of the count produced by each of said photodetectors and the count produced by one of said photodetectors selected as a reference, so that multiplying any subsequent measurement of luminescence by the inverse of the counting ratios computed for the respective photodetectors makes the measurement independent of the photodetector performing the counting.

15. A method for normalizing two or more photodetectors in a single-photon counting system for measuring the luminescence of multiple samples, said photodetectors comprising a reference detector and one or more other photodetectors, said method comprising:

successively counting the photon emissions of two or more standards with varying activity levels by two or more photodetectors, at least one of said photodetectors being the reference detector;

computing one or more counting ratios for each of said one or more other photodetectors, said one or more counting ratios being defined as the ratio of counts from the corresponding one or more other photodetectors of a standard to the counts from the reference detector of the same standard; and

in subsequent use of the photodetection system, multiplying the counts of a luminescent sample measured by a particular photodetector by the inverse of one of said one or more counting ratios computed for that particular photodetector, said one of said one or more counting ratios constituting a photodetector'"'"'s corresponding counting ratio.

16. The method of claim 15 wherein an average counting ratio for each of said one or more other photodetectors is computed from counts of said two or more standards with varying activity levels by each of said one or more other photodetectors,

and wherein said average counting ratio for each of said one or more other photodetectors constitutes each photodetector'"'"'s said corresponding counting ratio.

17. The method of claim 15 wherein a multitude of counting ratios for each of said one or more other photodetectors are computed from counts of said two or more standards with varying activity levels by each of said one or more other photodetectors, said multitude of counting ratios corresponding to the varying activity levels of the standards measured,

and wherein in subsequent use of the photodetection system, a photodetector'"'"'s corresponding counting ratio is chosen based on the activity level of the sample being measured from the set of said multitude of counting ratios for each of said one or more other photodetectors.

18. The method of claim 17 wherein the counting ratio chosen as a photodetector'"'"'s corresponding counting ratio is the counting ratio for the photodetector calculated from the measurement of the standard with an activity level closest to that of the sample being measured.

19. The method of claim 17 wherein for each of said one or more photodetectors a counting ratio-activity level equation is derived from the data obtained by the counting of the two or more standards with varying activity levels, and

wherein the counting ratio chosen as a photodetector'"'"'s corresponding counting ratio is the counting ratio for the photodetector calculated from that photodetector'"'"'s counting ratio-activity level equation based on the activity level of the sample being measured.

20. An apparatus for normalizing two or more photodetectors in a single-photon counting system for measuring the luminescence of multiple samples, said photodetectors comprising a reference detector and one or more other photodetectors, said apparatus comprising:

means for successively counting the photon emissions of a standard by two or more photodetectors, at least one of said photodetectors being the reference detector;

means for computing a counting ratio for each of said one or more other photodetectors, said counting ratio being defined as the ratio of counts from the corresponding one or more other photodetectors to the counts from the reference detector; and

means for multiplying the counts of a luminescent sample subsequently measured by a particular photodetector by the inverse of said counting ratio computed for that particular photodetector.

21. The apparatus of claim 20 wherein the photodetectors are photomultiplier tubes.

22. The apparatus of claim 20 wherein the standard comprises:

a scintillator with a peak wavelength approximately the same as that of said luminescent sample and a long decay constant,

one or more radioactive materials,

and a containment means.

23. The apparatus of claim 22 wherein said scintillator comprises calcium tungstate:

lead.

24. A standard for normalizing two or more photodetectors in a single-photon counting system used to measure the luminescence of samples, said standard comprising:

a scintillator whose photon emission has a peak wavelength of about 433 nanometers,

a radioactive material whose radioactive decay excites photon emission from the scintillator, and

containment means.

25. The standard of claim 24 wherein said radioactive material is either tritium or carbon-14 thymidine.

26. A standard for normalizing two or more photodetectors in a single-photon counting system used to measure the luminescence of samples, said standard comprising:

a scintillator whose photon emission has a peak wavelength of about 433 nanometers,

means for exciting said scintillator, and

a microplate.

27. A standard for normalizing two or more photodetectors in a single-photon counting system used to measure the luminescence of samples, said standard comprising:

a scintillator whose photon emission has a peak wavelength of about 433 nanometers,

means for exciting said scintillator,

containment means, and

means for maintaining said scintillator in said containment means, said maintaining means including a binding material composed of carboxy methyl cellulose.

28. A standard for normalizing two or more photodetectors in a single-photon counting system used to measure the luminescence of samples, said standard comprising:

a scintillator whose photon emission has a peak wavelength of about 433 nanometers,

means for exciting said scintillator,

containment means, and

means for maintaining said scintillator and said means for exciting said scintillator in said containment means, said maintaining means including a potting material.

29. The standard of claim 28 wherein, said potting material is an epoxy.

30. The standard of claim 28 wherein, said potting material is styrene.

31. A standard for normalizing two or more photodetectors in a single-photon counting system used to measure the luminescence of samples, said standard comprising:

a scintillator whose photon emission has a peak wavelength of about 433 nanometers, said scintillator comprising calcium tungstate;

lead,

means for exciting said scintillator, said exciting means being either tritium or carbon-14 thymidine,

a microplate,

means for maintaining said scintillator in said microplate, said means for maintaining said scintillator in said microplate comprising carboxy methyl cellulose, and

means for maintaining said scintillator and said means for exciting said scintillator in said microplate, said means for maintaining said scintillator and said means for exciting said scintillator in said microplate comprising an epoxy,