Modular fiber optic fluorometer and method of use thereof

• US 5,994,707 A
• Filed: 03/18/1997
• Issued: 11/30/1999
• Est. Priority Date: 03/18/1997
• Status: Expired due to Term

• Current Assignee: Physical Optics Corporation
• Original Assignee: Physical Optics Corporation
• Inventors: Forrester, Thomas C., Sorenson, James E., Lieberman, Robert A., Mendoza, Edgar A.
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US08/819,050
• Time in Patent Office: 987 Days
• Field of Search: 250/458.1, 250/459.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 2201/0221 Portable; cableless; compact; hand-held
• CPC Class Codes: G01N 2201/0245 with insertable-removable part
• CPC Class Codes: G01N 2201/086 Modular construction, e.g. disconnectable fibre parts

Abstract

A low cost portable fiber optic fluorometer is packaged as a personal computer peripheral and is based on interchangeable modules. One embodiment of the fluorometer comprises an excitation source, a detector, a signal processor, frequency source, wavelength selector, and an optical interface. The optical interface is exclusively fiber optic-based, thereby simplifying optical alignment and reducing the cost of the fluorometer. In another embodiment, the excitation source is an inexpensive monochromatic excitation source. In this case, the monochromatic excitation source and the first wavelength selector are preferably removable and replaceable, so that the fluorometer is advantageously able to generate different excitation wavelengths and detect different emission wavelengths. A fluorescence measurement method comprises the steps of generating an excitation signal; transmitting the excitation signal to a system under study which, in response, generates an emission signal; and detecting both the excitation signal and the emission signal with the fluorometer, and then using the detected excitation signal as a reference signal to calibrate the fluorometer and to nullify distortion errors in the emission signal. The use of the excitation signal as a reference signal provides an advantageously simple way to calibrate the fluorometer and to nullify distortion errors in the emission signal. An alternative method allows the precise shapes of the emission signal and the excitation signal to be profiled. The fluorometry method and apparatus can both advantageously be used for performing both direct fluorescence lifetime measurements and for performing phase fluorometry.

Claims

1. A fluorescence spectroscopy method comprising the steps of:

(A) venerating an excitation signal at a fluorometer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said excitation signal and said emission signal from said system under study to said fluorometer; and

(C) detecting said excitation signal and said emission signal with said fluorometer, said detected excitation signal and said detected emission signal both being received from said system under study;

(D) using said detected excitation signal as a reference signal to calibrate said fluorometer and to nullify distortion errors in said detected emission signal; and

(E) processing said emission signal to produce timing information, said processing step including the steps of

(1) detecting when said emission signal crosses a predetermined level;

(2) initiating a first delay when said emission signal crosses said predetermined level;

(3) detecting when a coarse counter changes state;

(4) initiating a second delay when said coarse counter changes state;

(5) causing said first delay and said second delay to terminate approximately simultaneously by adjusting the duration of at least one of said first and second delays;

(6) obtaining said timing information, including

(a) reading the value of said coarse counter to obtain relatively low precision timing information,

(b) determining the duration of said first and second delays to obtain relatively high precision timing information, and

(c) combining said high precision timing information with said low precision timing information.

2. A fluorescence spectroscopy method comprising the steps of:

(A) generating an excitation signal at a fluorometer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said excitation signal and said emission signal from said system under study to said fluorometer; and

(C) detecting said emission signal with said fluorometer, said detected emission signal being received from said system under study; and

(D) processing said emission signal to produce timing information, said processing step including the steps of

(1) detecting when said emission signal crosses a predetermined level;

(2) detecting when a counter changes state;

(3) generating a difference signal based on the difference in time between when said emission signal crosses said predetermined level and when said counter changes state;

(4) iteratively adjusting an adjustable oscillator based on said difference signal, said adjustable oscillator driving state changes of said counter, and said adjustable oscillator being iteratively adjusted until said emission signal crosses said predetermined level at approximately the same time as when said counter changes state; and

(5) obtaining said timing information by determining the frequency of said adjustable oscillator and by determining the value of said counter at least when said iteratively adjusting step is complete.

3. A fluorescence spectroscopy method comprising the steps of:

(A) generating an excitation signal at a fluorometer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said excitation signal and said emission signal from said system under study to said fluorometer; and

(C) detecting said emission signal with said fluorometer, said detected emission signal being received from said system under study; and

(D) processing said emission signal to produce timing information, said processing step including the steps of

(1) detecting when said emission signal crosses a predetermined level;

(2) obtaining said timing information based on when said emission signal crosses said predetermined level; and

(3) adjusting said predetermined level and repeating said detecting and obtaining steps, said adjusting and repeating steps being performed a plurality of times so as to obtain a plurality of data points which profile the exponential decay of said emission signal.

4. The method according to claim 3, further comprising the steps of

providing said fluorometer, said fluorometer comprising (i) an oscillator;

(ii) a excitation source which is coupled to said oscillator and which generates said excitation signal;

(iii) a wavelength selector which receives said emission signal from said system under study and which filters out components of light received from said system under study which are not components of said emission signal; and

(iv) a detector which is coupled to said wavelength selector and which detects said emission signal;

wherein said excitation source is provided as a modular excitation source which is removable and replaceable with a plurality of additional excitation sources such that said fluorometer is operable to generate a plurality of additional excitation signals having different excitation wavelengths, depending on the excitation source used;

wherein said excitation source and said plurality of additional excitation sources are each substantially monochromatic, such that an operating range of said fluorometer at a given instant is limited to a specific narrow range of wavelengths corresponding to the excitation source used.

5. The method according to claim 4 wherein, during said providing step, said fluorometer either (i) is packaged on an expansion card which is removably insertable into an expansion slot of a personal computer, or (ii) is packaged as a stand-alone peripheral for a personal computer and has a size which is not substantially larger than that of said personal computer.

6. The method according to claim 5, wherein the generating, detecting and processing steps are all performed using exclusively solid-state components.

7. A fluorescence spectroscopy method comprising the steps of:

(A) generating an excitation signal at a fluorometer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said excitation signal and said emission signal from said system under study to said fluorometer; and

(C) detecting said emission signal with said fluorometer, said detected emission signal being received from said system under study; and

(D) providing said fluorometer with automatic gain control circuitry; and

(E) holding pulse shapes of said emission signal substantially constant using said automatic gain control circuitry.

8. A fluorescence spectroscopy method comprising the steps of:

(A) generating an excitation signal at a fluorometer, said fluorometer either (i) being packaged on an expansion card which is removably insertable into an expansion slot of a personal computer, or (ii) being packaged as a stand-alone peripheral for a personal computer and has a size which is not substantially larger than that of said personal computer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said emission signal from said system under study to said fluorometer;

(C) detecting said emission signal with said fluorometer;

(D) processing said emission signal to produce timing information, said processing step including the steps of

(1) detecting when said emission signal crosses a predetermined level;

(2) obtaining said timing information based on when said emission signal crosses said predetermined level; and

(3) adjusting said predetermined level and repeating said detecting and obtaining steps, said adjusting and repeating step being performed a plurality of times so as to obtain a plurality of data points which profile the exponential decay of said emission signal.

9. The method according to claim 8,

wherein said generating step is performed using a solid-state modular excitation source which is removable and replaceable with a plurality of additional excitation sources such that said fluorometer is operable to generate a plurality of additional excitation signals having different excitation wavelengths, depending on the excitation source used, and

wherein said excitation source and said plurality of additional excitation sources are each substantially monochromatic, such that an operating range of said fluorometer at a given instant is limited to a specific narrow range of wavelengths corresponding to the excitation source used.

10. The method according to claim 9,

wherein said transmitting step is performed using an optical interface which interfaces said fluorometer with said system under study, and

wherein said optical interface is exclusively fiber optic-based.

11. The method according to claim 10, wherein the generating detecting and processing steps are all performed using exclusively solid-state components.

12. The method according to claim 11, further comprises the steps of

detecting said excitation signal after said excitation signal has returned from said system under study;

and

using said detected excitation signal as a reference signal to calibrate said fluorometer and to nullify distortion errors in said detected emission signal.

13. A fluorometer comprising:

(A) a solid-state, modular, removable and replaceable excitation source, said excitation source being substantially monochromatic, and said excitation source generating an excitation signal which is provided to a system under study;

(B) a solid-state, modular, removable and replaceable wavelength selector, said wavelength selector receiving an emission signal generated by said system under study in response to said excitation signal and filtering out components of light received from said system under study which are not components of said emission signal;

(C) a detector, said detector being coupled to said wavelength selector, and said detector detecting said emission signal;

(D) a solid-state signal processor, said signal processor being coupled to said detector, and said signal processor processing said emission signal; and

(E) an optical interface, including

(1) a fiber optic output, said fiber optic output being coupled to said excitation source,

(2) a first optical fiber, said first optical fiber being coupled to said fiber optic output and delivering said excitation signal to said system under study,

(3) a fiber optic input, said fiber optic input being coupled to said detector, and

(4) a second optical fiber, said second optical fiber being coupled to said fiber optic input and delivering said emission signal from said system under study to said fiber optic input,

(F) a programmable crossing detector which enables said fluorometer to profile the exponential decay of said emission signal; and

wherein said optical interface is the only optical interface between said fluorometer and said system under study through which fluorometric signals are communicated, and wherein said optical interface is exclusively fiber optic-based.

14. The fluorometer according to claim 13,

wherein said detector detects both said emission signal and said excitation signal, said detected emission signal and said detected excitation signal both being received from said system under study;

wherein said fluorometer further comprises a signal processor which is coupled to said detector which processes said detected emission signal;

wherein said signal processor uses said detected excitation signal as a reference signal to calibrate said fluorometer and to nullify distortion errors in said detected emission signal.

15. A fluorometer comprising:

an excitation source for generating an excitation signal and transmitting said excitation signal to a system under study;

a wavelength selector for receiving an emission signal generated by said system under study in response to said excitation signal;

a detector coupled to said wavelength collector for detecting said emission signal;

a programmable crossing detector that enables the fluorometer to profile the exponential decay of said emission signal; and

a signal processor coupled to said detector for receiving and processing said emission signal.

16. A fluorometer according to claim 15, further including an optical interface between the fluorometer and said system under study through which fluorometric signals are communicated, said optical interface being exclusively fiber optic based.

17. A fluorescence spectroscopy method comprising the steps of:

(A) generating an excitation signal at a fluorometer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said excitation signal and said emission signal from said system under study to said fluorometer; and

(C) detecting said emission signal and said excitation signal with said fluorometer, said detected emission and said excitation signal being received from said system under study; and

(D) using a crossing detector to determine a phase difference between said excitation signal and said emission signal.

18. A method according to claim 17, further comprising the steps of,

(A) processing said emission signal and said excitation signal to produce timing information, said processing step including the steps of

(1) detecting a crossing when said emission signal crosses a predetermined level;

(2) initiating first and second delays, one of said first and second delays being initiated when said emission signal crosses said predetermined level;

(3) causing said first delay and said second delay to terminate approximately simultaneously by adjusting the duration of at least one of said first and second delays; and

(4) producing said timing information based on said adjusting step.

19. A method according to claim 18, further comprising the step of obtaining said timing information by determining a total elapsed time between said generation of said excitation signal and said detection of said crossing.

20. A method according to claim 17, further comprising the steps of,

(A) processing said emission signal to produce timing information, said processing step including the steps of

(1) determining a first instant in time at which a first event occurs, said first instant in time being determined by when said emission signal crosses a perdetermined level;

(2) determining a second instant in time at which a second event occurs, said second instant in time being adjustable; and

(3) causing said first event and said second events to occur substantially simultaneously by adjusting when said second instant in time occurs.

21. A fluorescence spectroscopy method comprising the steps of:

(A) generating an excitation signal at a fluorometer;

(B) transmitting said excitation signal to a system under study which, in response, generates an emission signal, and then transmitting said excitation signal and said emission signal from said system under study to said fluorometer; and

(C) processing said emission signal to produce timing information, said processing step including the steps of

(1) detecting when said emission signal crosses a predetermined level;

(2) obtaining said timing information based on when said emission signal crosses said predetermined level; and

(3) adjusting said predetermined level and repeating said detecting and obtaining steps, said adjusting and repeating steps being performed a plurality of times so as to obtain a plurality of data points which profile the exponential decay of said emission signal.