Method and appartatus for improved time-resolved fluorescence spectroscopy

• US 4,855,930 A
• Filed: 03/27/1987
• Issued: 08/08/1989
• Est. Priority Date: 03/27/1987
• Status: Expired due to Term

• Current Assignee: Marco Travia
• Original Assignee: Chimerix Incorporated
• Inventors: Guignon, Ernest F., Fernandez, Salvador M., Chao, Yong-Sheng
• Primary Examiner(s): Lall, Parshotam S.
• Assistant Examiner(s): Cosimano, Edward R.
• Application Number:
  US07/031,288
• Time in Patent Office: 865 Days
• Field of Search: 250/361 C, 250/458.1, 250/459.1, 356/300, 356/308, 356/318, 356/319, 356/346, 364/497, 364/498, 364/550, 364/554, 378/44, 378/45
• US Class Current: 702/32
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 2201/0697 Pulsed lasers
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures

Abstract

A method and apparatus for time-resolved fluorescence spectroscopy is described in which laser light from a single pulse is used to excite fluorescent photons in a sample, which fluorescence is detected by a PMT optimized for linearity and response time to produce photoelectrons which generate a current at the PMT anode. This current is discharged through an R/C network to produce a voltage amplitude waveform which is converted to an optical image, intensified, stored and digitized. The digitized version of the optical image is processed in a data processor to calculate the true fluorescence impulse response.

Claims

1. A method for fluorescent spectroscopy of material, comprising the steps of:

(a) exciting the material with a single pulse of light energy to induce fluorescence;

(b) detecting in a detector a signal corresponding to the fluorescence transient waveform induced by said single pulse, as distorted by said detector and said pulse, and generating an electrical signal F(t) corresponding to said distorted signal;

(c) detecting in said detector a signal, which corresponds to said single pulse, as distorted by the impulse response of said detector, and generating an electrical signal E(t);

(d) separately displaying an image of said waveforms F(t) and E(t);

(e) digitizing a predetermined number of data points on each such image, as digital numbers representing points on said waveforms;

(f) storing said numbers in memory as data points of E(t) and F(t); and

(g) calculating the true fluorescence impulse response waveform f(t) from the stored data point numbers.

2. The method of claim 1 wherein the images displayed in step (d) are intensified and then stored in an image storage device for subsequent digitizing.

3. The method of claim 2 wherein the detector comprises a photomultiplier tube and the waveforms are in the form of amplitude varying voltages provided at the output of the photomultiplier tube.

4. The method of claim 3 wherein the voltage is applied to a sweep circuit of a cathode ray tube for display as an image on the face thereof and said image is intensified and stored by a charge-coupled device.

5. The method of claim 1 wherein the true fluorescence impulse waveform is calculated by convoluting E(t) with a predetermined trial function f (t) having adjustable parameters and comparing the data points of the resulting convoluted F(t)_calc with data points corresponding to F(t) data points.

6. The method of claim 5 wherein the predetermined trial function curve F(t)_calc is determined by physico-chemical considerations of the known characteristics of the constituents in the material.

7. The method of claim 5 wherein the material comprises a plural number i of fluorescent species and the trial function is the sum of a plural number i, of exponential curves A₁ exp(-t/T₁)+A₂ exp(-t/T₂) --- A_i exp(-t/T_i).

8. The method of claim 5 wherein the parameters of the trial function curve F(t)_calc are interatively adjusted until a best fit is achieved between the trial function curve F(t)_calc and the stored curve F(t).

9. The method of claim 8 wherein a best fit occurs when the variance between F(t)_calc and F(t) is minimized when successive values of the chi-square function thereof differ by less than a predetermined amount.

10. The method of claim 5 wherein the material comprises a single fluorescent species, the trial function curve is an exponential curve in the form of Aexp(-t/T) wherein the parameter A is the amplitude and T is the fluorescent decay lifetime.

11. The method of claim 10 wherein the material is tagged with a know fluorescent specie and T is therefore known in advance and the parameter A is proportional to the concentration of the fluorescent species in the material.

12. The method of claim 10 wherein the material is tagged with at least two known fluorescent specie having known fluorescent decay lifetimes which are known in advance and the amplitude parameter is proportional to the concentration of each fluorescent specie in the material.

13. The method of claims 11 or 12 wherein the absolute concentration of the fluorescent specie is determined by comparing the calculated amplitude parameter with a curve stored in computer memory;

said curve having been obtained in advance by analyzing a series of samples of know concentration and generating a table of the amplitude parameter A versus actual concentration.

14. Apparatus for fluorescent spectroscopy of material, comprising:

(a) a stable source of light pulses for exciting the material with a single pulse of light energy to induce fluorescence;

(b) detector means for separately;

(i) detecting the fluorescence transient waveform induced by said single pulse, as distorted by said detector means and generating an electrical waveform signal F(t) corresponding thereto, and

(ii) detecting the waveform, which represent said single pulse, as distorted by the impulse response of said detector means and generating an electrical waveform signal E(t) corresponding thereto,

(c) image display means responsive to said electrical signals for separately displaying an image of said waveform signals F(t) and E(t);

(d) digitizing means for producing a predetermined number of data points on each such image, as digital numbers representing points on said waveform signals;

(e) storage means for storing said numbers in memory as data points of E(t) and F(t); and

(f) computer means for calculating the true fluorescence impulse response waveform f(t) from the stored data points.

15. The apparatus of claim 14 wherein the light source comprises a pulsed laser, the detector means comprises a photomultiplier tube for generating output electrical signals, the electrical signals are voltage amplitude waveforms, the image display means comprises a cathode ray tube having an input sweep circuit, which sweep circuit is coupled to the output electrical signals from said photomultiplier tube for displaying said voltage waveforms on the face of the cathode ray tube, and image intensifier means for intensifying the image on said face and an image storage device coupled to said intensifier means for storing said image as charges on an array of charge-coupled devices.

16. The apparatus of claim 14 including beam splitter means for sampling a portion of the light energy to generate control signal to maintain a constant light intensity input to the detector means.

17. The apparatus of claim 16 wherein the detector means comprises a photomultiplier tube, the light pulses comprise pulses of laser energy which induce fluorescence photons, said light energy is split by said beam splitter into a main beam and a sampling beam, said sampling beam is detected to generate a control signal which is used to control the position of a gradient density filter in the path of the main beam to maintain constant light intensity to the input of said photomultiplier tube.

18. The apparatus of claim 14 including means for calculating the true impulse response function convoluting E(t) with a predetermined trial function t(f) having adjustable parameters and comparing the data points of the convoluted F(t)_calc with data points corresponding to the F(t) data points.

19. The apparatus of claim 18 including means for adjusting the parameters of the trial function curve F(t)_calc until a best fit is achieved between the trial function curve F(t)_calc and the stored curve F(t).

20. The apparatus of claim 18 wherein the material comprises a plural number i of fluorescent species and the trial function is the sum of a plural number, i, of exponential curves A₁ exp(-t/T₁)+A₂ exp(-t/T₂) --- A_i exp(-t/T_i).

21. The apparatus of claim 18 wherein the material is tagged with at least two known fluorescent specie having known fluorescent decay lifetimes which are known in advance and the amplitude parameter is proportional to the concentration of each fluorescent specie in the material.

22. The apparatus of claim 18 wherein the material comprises a single fluorescent species, the trial function curve is an exponential curve in the form of Aexp(-t/T) wherein the parameter A is the amplitude and T is the fluorescent decay lifetime.

23. The apparatus of claim 22 wherein the material is tagged with a known flurescent specie and T is therefore known in advance and the parameter A is proportional to the concentration of the fluorescent species in the material.