Optoinjection methods

• US 6,753,161 B2
• Filed: 09/21/2001
• Issued: 06/22/2004
• Est. Priority Date: 03/27/1997
• Status: Expired due to Term

• Current Assignee: Intrexon Corporation
• Original Assignee: Oncosis, LLC
• Inventors: Palsson, Bernhard O., Hanania, Elie G., Eisfeld, Timothy M., Koller, Manfred R.
• Primary Examiner(s): Weber, Jon P.
• Assistant Examiner(s): Srivastava, Kailash C.
• Application Number:
  US09/961,691
• Publication Number:
  US 20020076744A1
• Time in Patent Office: 1,005 Days
• Field of Search: 435/40.5, 435/173.1, 435/244, 435/254, 435/255, 435/256, 435/288.7, 435/240.1, 435/240.2, 435/240.3, 435/240.4
• 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 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• 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 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• 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 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

Abstract

Optoinjection method for transiently permeabilizing a target cell by (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.

Claims

1. A method for transiently permeabilizing a target cell, comprising the steps of

(a) illuminating a population of cells contained in a frame;

(b) detecting at least one property of light directed from the frame;

(c) locating a target cell in the population of cells, wherein the target cell is located with reference to the detected property of light; and

(d) irradiating the target cell with a pulse of radiation, wherein the pulse of radiation has a diameter of at least 10 μ

m at the point of contact with the target cell;

whereby the target cell is transiently permeabilized.

3. The method of claim 1, wherein the population of cells is substantially stationary.

4. The method of claim 1, wherein the at least one property of light detected in step (b) is obtained as a static representation of light transmitted simultaneously from the frame, whereby the target cell located in step (c) is located with reference to the static representation.

5. The method of claim 1, wherein at least one property of light is fluorescence and the target cell is located with reference to the fluorescence.

6. The method of claim 1, wherein the population of cells is illuminated through a lens having numerical aperture of at most 0.5 and the target cell is located with reference to a property of light directed from the frame and through the lens.

8. The method of claim 1, wherein the pulse of radiation has a diameter of at least 20 μ

m at the point of contact with the target cell.

9. The method of claim 1, wherein the pulse of radiation delivers at most 2 μ

J/μ

m².

2. A method for transiently permeabilizing a target cell, comprising the steps of

(a) illuminating a population of cells contained in a frame;

(b) detecting at least one property of light directed from the frame;

(c) locating a target cell in the population of cells, wherein the target cell is located with reference to the detected property of light; and

(d) irradiating the target cell with a pulse of radiation, wherein the pulse of radiation delivers at most 2 μ

J/μ

m²;

whereby the target cell is transiently permeabilized.

7. The method of claim 2, wherein the pulse of radiation has a diameter of at least 5 μ

m at the point of contact with the target cell.

10. The method of claim 2, wherein the pulse of radiation delivers at most 0.1 μ

J/μ

m².

11. The method of claim 2, wherein the pulse of radiation delivers at most 0.01 μ

J/μ

m².

12. The method of claim 2, wherein the pulse of radiation delivers at most 1 μ

J/μ

m².

13. The method of claim 2, wherein the population of cells is substantially stationary.

14. The method of claim 2, wherein the at least one property of light detected in step (b) is obtained as a static representation of light transmitted simultaneously from the frame, whereby the target cell located in step (c) is located with reference to the static representation.

15. The method of claim 2, wherein at least one property of light is fluorescence and the target cell is located with reference to the fluorescence.

16. The method of claim 2, wherein the population of cells is illuminated through a lens having numerical aperture of at most 0.5 and the target cell is located with reference to a property of light directed from the frame and through the lens.

17. The method of claim 2, further comprising the step of (e) adjusting the direction of the pulse of radiation to irradiate a second target cell in the population, whereby the second target cell is transiently permeabilized.

18. The method of claim 2, wherein the frame has an area of at least 50 mm².

19. The method of claim 2, wherein the frame has an area of at least 85 mm².

20. The method of claim 2, wherein the frame has an area of at least 115 mm².

21. The method of claim 2, wherein the property of light is transmittance and the target cell is located with reference to the transmittance.

22. The method of claim 2, wherein the property of light is polarization and the target cell is located with reference to the polarization.

23. The method of claim 2, wherein the property of light is reflectance and the target cell is located with reference to the reflectance.

24. The method of claim 2, wherein the property of light is phase contrast illumination and the target cell is located with reference to the phase contrast illumination.

25. The method of claim 2, wherein the target cell is a procaryotic cell.

26. The method of claim 25, wherein said procaryotic cell is a bacterial cell.

27. The method of claim 2, wherein the target cell is a eucaryotic cell.

28. The method of claim 2, wherein the target cell is selected from the group consisting of an animal cell, plant cell, yeast cell, human cell and non-human primate cell.

29. The method of claim 2, wherein the population of cells contains cells associated with an exogenous label.

30. The method of claim 29, wherein the label is a fluorophore.

31. The method of claim 2, wherein the target cell is associated with an exogenous label.

32. The method of claim 2, wherein the target cell is in the presence of an exogenous molecule.

33. The method of claim 32, wherein the exogenous molecule is selected from the group consisting of a nucleic acid, polypeptide, carbohydrate, lipid, and small molecule.

34. The method of claim 33, wherein the small molecule is a dye capable of absorbing visible, ultraviolet or infrared light.

35. The method of claim 14, wherein step (b) further comprises obtaining a second static representation of at least one property of light directed simultaneously from the frame.

36. The method of claim 35, wherein the target cell is located with reference to the first and second static representations.

37. The method of claim 2, further comprising:

(e) locating additional target cells in the population of cells, wherein the target cells are located with reference to the detected property of light, and

(f) irradiating each of the target cells with a pulse of radiation, wherein the pulse of radiation has a diameter of at least 10 μ

m at the point of contact with each target cell;

whereby additional target cells are transiently permeabilized.

38. The method of claim 37, wherein at least 10,000 cells are irradiated per minute.

40. The method of claim 37, wherein at least 50,000 cells are irradiated per minute.

41. The method of claim 37, wherein at least 100,000 cells are irradiated per minute.

42. The method of claim 2, further comprising the steps of

(e) illuminating a population of cells contained in a second frame,

(f) obtaining a static representation of at least one property of light directed from the second frame, and repeating steps (c) through (d).

43. The method of claim 42, wherein the population of cells remains in a substantially stationary location relative to a lens.

44. The method of claim 42, wherein at least 10,000 cells are irradiated per minute.

46. The method of claim 42, wherein at least 50,000 cells are irradiated per minute.

47. The method of claim 42, wherein at least 100,000 cells are irradiated per minute.

48. The method of claim 42, further comprising the step of

(g) moving the population of cells and repeating steps (a) through (f).

49. The method of claim 42, wherein steps (a) through (f) are automated.

50. The method of claim 2, further comprising the steps of

(e) moving the population of cells relative to a lens and repeating steps (a) through (d).

51. The method of claim 2, wherein steps (a) through (d) are automated.

52. The method of claim 14, wherein the static representation comprises an image.

53. The method of claim 14, wherein the static representation comprises a set of data stored in computer memory.

54. The method of claim 2, further comprising a camera having a magnification between 2×

and 40×

.

55. The method of claim 2, further comprising a camera having a magnification between 2.5×

and 25×

.

56. The method of claim 16, wherein the lens has a numerical aperture of at most 0.3.

57. The method of claim 37, wherein the property of light is intensity and the target cell is located with reference to the intensity.

58. The method of claim 37, wherein greater than 50% of the irradiated target cells are viable after the method is performed.

59. The method of claim 37, wherein greater than 80% of the irradiated target cells are viable after the method is performed.

60. The method of claim 37, wherein greater than 90% of the irradiated target cells are viable after the method is performed.

39. The method of 37, wherein at least 20,000 cells are irradiated per minute.

45. The method of 42, wherein at least 20,000 cells are irradiated per minute.