Apparatus for eliminating background interference in fluorescence measurements

• US 4,573,796 A
• Filed: 01/06/1984
• Issued: 03/04/1986
• Est. Priority Date: 01/06/1984
• Status: Expired due to Fees

• Current Assignee: United States Department of Energy, Regents of the University of California (University of California)
• Original Assignee: United States Department of Energy
• Inventors: Martin, John C., Jett, James H.
• Primary Examiner(s): McGraw, Vincent P.
• Assistant Examiner(s): Thompson, III, Robert D. V.
• Application Number:
  US06/568,768
• Time in Patent Office: 788 Days
• Field of Search: 356/318, 356/317, 356/73, 356/39, 356/417, 356/343, 250/458.1, 250/459.1, 250/461.1, 250/461.2
• US Class Current: 356/318
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 2015/1438 Using two lasers in succession
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• 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 21/645 Specially adapted constructive features of fluorimeters

Abstract

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle.

Claims

1. In a flow cytometer measuring device having first and second light sources for forming respective first and second beams of focused light to excite different fluorescent dyes bound to specific components of biological particles, the measuring device including a fluorescence detector for producing fluorescence data, and a data storage system for storing said fluorescence data, the improvement comprising an apparatus for modulating on and off light from a light source to eliminate background interference from one light source while the other light source excites the biological particle and the resulting fluorescence is detected, said apparatus including:

a. first and second light scatter detectors for detecting light scattered by biological particles;

b. first and second gate signal generators for generating first and second gate signals, said first gate signal generator being responsive to said first light scatter detector and said second gate signal generator being responsive to said second light scatter detector;

c. first and second gated signal processing means for receiving said first and second gate signals respectively and for transmitting the fluorescence data to the data storage system, said first gated signal processing means being connected to said first gate signal generator, the fluorescence detector and the data storage system, said second gated signal processing means being connected to said second gate signal generator, the fluorescence detector and the data storage system;

d. delay means for delaying said gate signal generated by one of said gate signal generators, said delay means being connected to receive said gate signal and producing a delayed gate signal in response thereto; and

e. an optical modulator associated with one of the light sources, said optical modulator being connected to said delay means to receive said delayed gate signal and modulate on and off the light from the light source in response thereto.

2. The invention of claim 1 wherein the light sources are lasers.

3. The invention of claim 1 wherein the biological particles are chromosomes.

4. The invention of claim 1 wherein each light source emits light having the same wavelength.

5. The invention of claim 1 wherein each light source emits light having different wavelengths.

6. The invention of claim 1 wherein the flow cytometer measuring device includes first and second fluorescence detectors, said first gated signal processing means being connected to the first fluorescence detector and said second gated signal processing means being connected to the second fluorescence detector.

7. The invention of claim 1 wherein said optical modulator is associated with the second light source and wherein said delay means delays said first gate signal generated by said first gate signal generator until the biological particle has moved from the first light beam to the second light beam.

8. The invention of claim 1 wherein said optical modulator is associated with the first light source and wherein said optical modulator modulates off light from the first light source after the first light scatter detector detects a biological particle until the same biological particle is detected by the second light scatter detector.

9. The invention of claim 8 further comprising a second optical modulator, said second optical modulator being associated with the second light source and wherein said delay means delays said first gate signal generated by said first gate signal generator until the biological particle has moved from the first light beam to the second light beam.

10. In a flow cytometer measuring device having first and second light sources for forming respective first and second beams of focused light to excite different fluorescent dyes bound to specific components of sufficiently small biological particles that light of sufficient strength to be detected is not scattered, the measuring device including first and second fluorescence detectors for producing fluorescence data, and a data storage system for storing said fluorescence data, the improvement comprising an apparatus for modulating on and off light from a light source to eliminate background interference from one light source while the other light source excites the biological particle and the resulting fluorescence is detected, said apparatus including:

a. first and second gate signal generators for generating first and second gate signals, said first gate signal generator being responsive to the first fluorescence detector and said second gate signal generator being responsive to the second fluorescence detector;

b. first and second gated signal processing means for receiving said first and second gate signals respectively and for transmitting the fluorescence data to the data storage system, said first gated signal processing means being connected to said first gate signal generator, the first fluorescence detector and the data storage system, said second gated signal processing means being connected to said second gate signal generator, the second fluorescence detector and the data storage system;

c. delay means for delaying the gate signal generated by one of said gate signal generators, said delay means being connected to receive said gate signal and producing a delayed gate signal in response thereto; and

d. an optical modulator associated with one of the light sources, said optical modulator being connected to said delay means to receive said delayed gate signal and modulate on and off the light from the light source in response thereto.

11. The invention of claim 10 wherein the biological particles are chromosomes.

12. The invention of claim 10 wherein the light sources are lasers.

13. The invention of claim 10 wherein each light source emits light having the same wavelength.

14. The invention of claim 10 wherein each light source emits light having different wavelengths.