Method and apparatus for selectively targeting specific cells within a cell population

• US 6,514,722 B2
• Filed: 11/30/2000
• Issued: 02/04/2003
• Est. Priority Date: 03/27/1997
• Status: Expired due to Term

• Current Assignee: Intrexon Corporation
• Original Assignee: Oncosis, LLC
• Inventors: Palsson, Bernhard O., Eisfeld, Timothy M., Koller, Manfred R.
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US09/728,281
• Publication Number:
  US 20010005586A1
• Time in Patent Office: 796 Days
• Field of Search: 435/40.5, 435/6, 435/7.1, 435/14, 435/29
• US Class Current: 435/40.5
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 5/0087 Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: C12N 5/0093 Purging against cancer cells
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 2021/1725 Modulation of properties by light, e.g. photoreflectance
• 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
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: Y10S 128/922 including image analysis

Abstract

This invention provides a method and apparatus for selectively identifying, and targeting with an energy beam, specific cells within a cell population, for the purpose of inducing a response in the targeted cells. Using the present invention, every detectable cell in a population can be identified and affected, without substantially affecting non-targeted cells within the mixture.

Claims

1. A method for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

illuminating a frame of cells within a field-of-view in said biological specimen, wherein said field-of-view comprises a plurality of frames of cells;

capturing an image of said frame of cells;

determining the locations of said one or more targeted cells within said image; and

steering an energy beam to the locations of said one or more targeted cells, wherein said energy beam is sufficient to induce a response in at least one of said one or more targeted cells.

2. The method of claim 1, wherein determining the locations of said one or more targeted cells comprises determining the presence of, or absence of, a label attached to said one or more targeted cells.

3. The method of claim 1, wherein capturing an image of said frame of cells comprises capturing an image of labeled cells.

4. The method of claim 1, wherein illuminating said frame of cells comprises illuminating said frame of cells with an excitation laser light source.

5. The method of claim 1, wherein said response is selected from the group consisting of cell death, optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, excitation of a fluorescent reagent, photobleaching and internal molecular uncaging.

6. The method of claim 1, wherein said response is non-lethal.

7. The method of claim 6, wherein said response is selected from the group consisting of optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, and excitation of a fluorescent reagent, photobleaching and internal molecular uncaging.

18. The method of claim 7, wherein said response is selected from the group consisting of optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, excitation of a fluorescent reagent, photobleaching and internal molecular uncaging.

8. A method for rapidly inducing a response in individual cells within a cell population, said cell population being held in a container, comprising:

illuminating a first frame of cells in a first field-of-view in said cell population;

wherein said field of view comprises a plurality of frames of cells;

capturing an image of said first frame of cells within said first field-of-view;

determining the locations of first individual cells within said first frame of cells by reference to said image; and

steering an energy beam to the locations of said first individual cells, wherein said energy beam is sufficient to induce a response in at least one of said first individual cells.

9. The method of claim 8, further comprising:

capturing an image of a second frame of cells within said first field-of-view;

determining the locations of second individual cells within said second frame of cells by reference to said image; and

steering an energy beam to the locations of said second individual cells within said second frame of cells, wherein said energy beam is sufficient to induce a response in at least one of said second individual cells.

10. The method of claim 8, further comprising:

illuminating a first frame of cells in a second field-of-view in said cell population;

capturing an image of said first frame of cells within said second field-of-view;

determining the locations of third individual cells within said first frame of cells within said second field-of-view by reference to said image; and

steering an energy beam to the locations of said third individual cells, wherein said energy beam is sufficient to induce a response in at least one of said third individual cells.

11. The method of claim 10, wherein illuminating said first frame of cells in said second field-of-view comprises moving said container and illuminating said first frame of cells.

12. The method of claim 10, wherein said response is selected from the group consisting of cell death, optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, excitation of a fluorescent reagent, photobleaching and internal molecular uncaging.

13. The method of claim 10, wherein said response is non-lethal.

14. The method of claim 13, wherein said response is selected from the group consisting of optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, excitation of a fluorescent reagent, photobleaching and internal molecular uncaging.

15. A method for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

focusing a scanning lens on a field-of-view within said biological specimen, said field-of-view comprising a plurality of frames of cells;

illuminating one or more frames of cells within said field-of-view;

capturing an image of a first frame of cells in said field-of-view;

determining the locations of said one or more targeted cells in said first frame of cells;

emitting an energy beam sufficient to induce a response in at least one of said one or more targeted cells; and

steering said energy beam to the locations of said one or more targeted cells within said first frame of cells.

16. The method of claim 15, wherein said response is selected from the group consisting of cell death, optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, and excitation of a fluorescent reagent.

17. The method of claim 15, wherein said response is non-lethal.

19. The method of claim 15, wherein capturing an image of said first frame of cells comprises capturing an image of labeled cells.

20. The method of claim 15, wherein said illuminating one or more frames of cells comprises illuminating said one or more frames of cells with an excitation laser light source.