Method and apparatus for measuring fluorescence

• US 5,990,484 A
• Filed: 10/14/1998
• Issued: 11/23/1999
• Est. Priority Date: 10/29/1997
• Status: Expired due to Fees

• Current Assignee: Laboratory Of Molecular Biophotonics
• Original Assignee: Laboratory Of Molecular Biophotonics
• Inventors: Ohsuka, Shinji
• Primary Examiner(s): EVANS, FANNIE L
• Application Number:
  US09/170,998
• Time in Patent Office: 405 Days
• Field of Search: 356/317, 356/318, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/461.2
• US Class Current: 250/458.1
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence

Abstract

The present invention relates to a method and apparatus for efficiently measuring the lifetime of the fluorescence emitted from a fluorescent material in response to pulsed light. The measuring apparatus according to the present invention includes, at least, an excitation light source whose excitation power is regulated such that at least one fluorescence photon can be detected per light pulse on average. While repeatedly irradiating a sample containing the fluorescent material with excitation pulses of light from the excitation light source, a fluorescence detection time from irradiation to fluorescence detection and the number of detected fluorescence photons are measured for each excitation pulse of light. Then, on the basis of a data group composed of pairs of the measurement data of fluorescence photon detection time and fluorescence photon number, both of which are measured for each occurrence of excitation pulse of light, the lifetime and fluorescence decay curve of the fluorescence emitted from the fluorescent material are determined.

Claims

1. A method of determining lifetime of fluorescence emitted from a fluorescent material contained in a sample by use of pulsed excitation light, said method comprising:

repeatedly irradiating a sample with pulses of excitation light;

measuring, for each occurrence of irradiation by a pulse of excitation light, a fluorescence detection time extending from when the sample is irradiated with a pulse of excitation light until a fluorescence photon from the fluorescent material excited by the excitation pulse of light is detected, and the number of fluorescence photons detected; and

determining at least the lifetime of the fluorescence emitted from the fluorescent material from a measurement data group measured for each occurrence of irradiation by an excitation pulse of light, the measurement data group including data concerning the fluorescence photon detection time and data concerning the number of fluorescence photons, both measured for each occurrence of irradiation by an excitation pulse of light.

2. The method according to claim 1, wherein the lifetime of the fluorescence emitted from the fluorescent material is determined, with reference to a likelihood which includes the measurement data and a variable yielding the lifetime of the fluorescence as an unknown parameter, from a value of the unknown parameter which maximizes the likelihood.

3. The method according to claim 1, wherein the excitation power of the excitation pulse of light reaching the sample is regulated so that at least one fluorescence photon can be detected per excitation light pulses, on average.

4. An apparatus for determining lifetime of fluorescence emitted from a fluorescent material contained in a sample by use of pulses of excitation light, said apparatus comprising:

an excitation light source for repeatedly irradiating a sample with pulses of excitation light;

a photodetector for detecting a fluorescence photon emitted from the fluorescent material, said photodetector comprising a photocathode for receiving the fluorescence emitted from the fluorescent material and releasing photoelectrons in a number corresponding to intensity of the emitted fluorescence, an electron multiplying unit for multiplying the photoelectrons released from said photocathode and releasing secondary electrons, a plurality of anode electrodes, each anode electrode capturing the secondary electrons arriving from said electron multiplying unit, and a vacuum envelope having an entrance window transmitting the fluorescence from the fluorescent material and containing said photocathode, said electron multiplying unit, and said plurality of anode electrodes;

a time measuring system for measuring, for each excitation pulse radiated by said excitation light source, a fluorescence photon detecting time extending from when the sample is irradiated with the excitation pulse of light until a fluorescence photon from the fluorescent material excited by the excitation pulse of light is detected;

a fluorescence photon number measuring system for measuring, for each excitation pulse of light radiated by said excitation light source, of said plurality of anode electrodes, the number of anode electrodes which have output an electric signal in response to a captured secondary electron as the number of fluorescence photons detected; and

a fluorescence lifetime determining system for determining at least the lifetime of the fluorescence emitted from the fluorescent material from a measurement data group measured for each occurrence of an irradiated pulse of light, said measurement data group including data concerning the fluorescence photon detection time and data concerning the number of fluorescence photons, both measured for each occurrence of a radiated excitation pulse of light.

5. The apparatus according to claim 4, wherein said fluorescence lifetime determining system determines at least the lifetime of the fluorescence emitted from the fluorescent material with reference to a likelihood which includes the measurement data and a variable yielding the lifetime of the fluorescence as an unknown parameter, from a value of the unknown parameter which maximizes the likelihood.

6. The apparatus according to claim 4, wherein said excitation light source regulates excitation power of the excitation pulses of light reaching the sample so that at least one fluorescence photon can be detected per excitation pulse of light, on average.

7. The apparatus according to claim 4, wherein said excitation light source regulates excitation power of the excitation pulses of light reaching the sample so that the fluorescence photons detected per excitation pulse of light does not exceed the number of said anode electrodes.