Method and apparatus for the measurement of low-level laser-induced fluorescence

• US 4,758,727 A
• Filed: 02/12/1986
• Issued: 07/19/1988
• Est. Priority Date: 02/12/1986
• Status: Expired due to Fees

• Current Assignee: Ohio State University Research Foundation A Instrumentality of Ohio
• Original Assignee: The Ohio State University Research Foundation
• Inventors: Tomei, L. David, Boninger, Michael, Jagadeesh, Jogikal, Cornhill, Fred
• Primary Examiner(s): Howell, Janice A.
• Assistant Examiner(s): HANIG, RICHARD E
• Application Number:
  US06/828,694
• Time in Patent Office: 888 Days
• Field of Search: 250/458.1, 250/459.1, 250/461.2, 250/461.1, 350/96.27, 350/527
• US Class Current: 250/458.1
• CPC Class Codes: G01J 2001/0481 Preset integrating sphere or cavity
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/6402 Atomic fluorescence; Laser induced fluorescence
• CPC Class Codes: G01N 21/643 non-biological material
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 2201/0833 Fibre array at detector, resolving

Abstract

A method and apparatus for measuring low-level laser-induced fluorescence are disclosed. A laser is used to produce a coherent beam of light which is sequentially passed through a beam expander, iris diaphram, focusing lens and three-dimensional scanner before being focused onto a rigidly mounted target. A computer is used to predeterminately control the pattern and the rate at which the scanner passes the beam of light over the target. The light transmitted onto the target induces fluorescent light in the target. The fluorescent light is sequentially gathered by a biased cut optical fiber member and directed into a photomultiplier tube where the intensity of the fluorescent light is measured. The intensity data is then digitized and recorded by the computer as a function of the coordinates of each preprogrammed point location of the beam impinging upon the target. This data is used to produce an image of all or a portion of the target on a visual monitor. In addition, the data can be recalled and used for further analysis of the target.

Claims

1. An apparatus for measuring the excited fluorescence of an object comprising:

means for generating a coherent beam of light;

a three dimensional scanner having means for securing such object in a fixed position, said scanner receiving and passing said coherent beam of light onto such fixed object in a desired pattern and at a desired rate, said scanner having X, Y coordinate scanning capability with simultaneous Z coordinate correction to cause said beam to have a topographical field of focus in the place of said object;

means for controlling the pattern and rate at which said beam of light is passed onto said object;

an optical fiber member for gathering and directing the fluorescent light induced from said object by said beam of light and for attenuating any direct light received by said object;

means for collecting and measuring the intensity of the fluorescent light received from said object; and

,

means for sequentially recording data regarding the intensity of the fluorescent light received from said object as a function of the location of said scanning beam of light on said object by said scanner.

2. The apparatus of claim 1 further including a means for filtering said fluorescent light emanating from said optical fiber member.

3. The apparatus of claim 1 further including a means for diffusing said fluorescent light emanating from said optical fiber member.

4. The apparatus of claim 1, further including means for producing a visual image of at least a portion of said object based upon said sequentially recorded data of the intensity of said transmitted light as a function of the location of said scanning beam of light on said object.

5. The apparatus of claim 1, which further includes a focusing lens interposed between said means for generating a coherent beam of light and said three dimensional scanner.

6. The apparatus of claim 5, which further includes a beam expander interposed between said means for generating a coherent beam of light and said focusing lens.

7. The apparatus of claim 6, which further includes an iris diaphram interposed between said beam expander and said focusing lens.

8. The apparatus of claim 1, wherein said means for diffusing said transmitted light comprises an open-ended hollow elongated member having a reflective interior surface.

9. The apparatus of claim 1, wherein said means for collecting and measuring the intensity of the light transmitted through said translucent object comprises a photomultiplier tube.

10. The apparatus of claim 1, wherein said means for predeterminately controlling the pattern and rate at which said beam of light is passed over said object is a programmable computer.

11. The apparatus of claim 1, wherein said means for sequentially recording data is a programmable computer.

12. An apparatus for measuring the excited fluorescence of an object having a binding fluorescent dye application comprising:

means for generating a coherent beam of light;

means for expanding said coherent beam of light;

means for focusing said expanded beam of light;

a three dimensional scanner having means for securing such object in a fixed position, said scanner receiving and passing said focused beam of light over such fixed object in a selected pattern and at a selected rate, said scanner having X, Y coordinate scanning capability with simultaneous Z coordinate correction to cause said focused beam of light to have a topographical field of focus in the plane of said object;

means for controlling the pattern and rate at which said beam of light is passed through said translucent object;

a biased cut optical fiber faceplate for receiving and directing the fluorescent light transmitted from said object;

means for filtering said fluorescent light emanating from said fiber faceplate;

means for diffusing said filtered light;

means for collecting and measuring the intensity of the fluorescent light transmitted through said object; and

means for sequentially recording data regarding the intensity of the fluorescent light transmitted from said object as a function of the location of said scanning beam of light on said object.

13. The apparatus of claim 12, which further includes means for producing a visual image of at least a portion of said object based upon said sequentially recorded data as a function of the location of the fluorescent light transmitted from said object.

14. The apparatus of claim 12, which further includes an iris diaphram interposed between said means for expanding said beam of light and said means for focusing said expanded beam of light.

15. The apparatus of claim 12, wherein said means for generating a coherent beam of light is a laser.

16. The apparatus of claim 12, wherein said means for expanding said beam of light is a variable beam expander.

17. The apparatus of claim 12, wherein said means for controlling the pattern and rate at which said beam of light is passed over said object is a programmable computer.

18. The apparatus of claim 12, wherein said means for diffusing said fluorescent light is an open-ended hollow elongated member having a reflective interior surface.

19. The apparatus of claim 12, wherein said means for collecting and measuring the intensity of the fluorescent light transmitted from said translucent object is a photomultiplier tube.

20. The apparatus of claim 12, wherein the means for sequentially recording data is a programmable computer.

21. An apparatus for measuring the excited fluorescence of an object having a finding fluorescent dye application comprising;

a laser for generating a coherent beam of light at a specified wavelength with the range of 350 to 540 nanometers;

a variable beam expander for expanding said coherent beam of light;

a focusing lens to focus said expanded beam of light;

a three dimensional scanner having means for securing such object in a fixed position, said scanner receiving and passing said focused beam of light over such fixed object in a selected pattern and at a selected rate, said scanner having X, Y coordinate scanning capability with simultaneous Z coordinate correction to cause said focused beam of light to have a topographical field of focus in the plane of said object;

means for controlling the pattern and rate at which said focused beam of light is passed over said object;

a biased cut optical fiber faceplate for receiving and directing the fluorescent light transmitted from said object;

filter means for receiving said fluorescent light from said fiber optic bundle and removing unwanted light input;

diffusion means for diffusing said filtered light;

a photomultiplier for measuring the intensity of the fluorescent light transmitted and received from said diffusion means; and

,

means for sequentially recording data regarding the intensity of the fluorescent light transmitted from said object as a function of the location of said scanning beam of light on said object.

22. The apparatus of claim 21, wherein said means for controlling the pattern and the rate at which said focused beam of light is passed over said object is a programmable computer.

23. The apparatus of claim 21, wherein said means for sequentially recording data is a programmable computer.

24. The apparatus of claim 21, wherein said diffusion means is an open-ended hollow elongated member having a reflective interior surface.

25. The apparatus of claim 21, which further includes an iris diaphragm interposed between said beam expander and said focusing lens.

26. The apparatus of claim 21, which further includes means for producing a visual image of at least a portion of said object based upon said sequentially recorded data as a function of the location of the fluorescent light transmitted from said object.