IN SITU ANALYSIS OF TISSUES

• US 20110190145A1
• Filed: 11/09/2010
• Published: 08/04/2011
• Est. Priority Date: 02/17/2005
• Status: Active Grant

• Current Assignee: Richard Caprioli
• Original Assignee: Richard Caprioli
• Inventors: Caprioli, Richard
• Granted Patent: US 8,304,196 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms

Abstract

The present invention provides for the simultaneous assessment of a plurality of tissue regions or microregions, the benefit being homogeneity of the sampling, both in terms of tissue content and timing. Discrete regions of a tissue sample, such as those demarcated by microwells formed within the tissue itself or tissue plugs removed from the tissue in a spatially referenced fashion, can be treated with one or more physical or chemical treatments to liberate target molecules of interest. Subsequent analysis of said target molecules by, e.g., mass spectroscopy, permits identification of a variety of biological parameters, including those associated with disease or therapy.

Claims

1. A method for analyzing protein content in a tissue comprising:

(a) providing an intact tissue comprising a first spatially discrete microregion;

(b) subjecting said microregion to one or more physical or chemical treatments; and

(c) analyzing a protein sample from said microregion, thereby providing analysis of protein content in said tissue.

2. The method of claim 1, wherein said intact tissue comprises at least a second spatially discrete microregion, and said method further comprises subjecting said second spatially discrete microregion to one or more physical or chemical treatments, and analyzing discrete protein samples from said second spatially discrete microregion.

3. The method of claim 2, wherein the protein content of said first and second spatially discrete microregions are compared.

4. The method of claim 1, wherein said tissue is an animal tissue.

5. The method of claim 4, wherein said animal tissue is heart tissue, liver tissue, kidney tissue, prostate tissue, breast tissue, ovary tissue, uterine tissue, skin tissue, lung tissue, brain tissue, colon tissue, head &

neck tissue, pancreatic tissue, muscle tissue or skin or tissue that contains body fluids or contains traces of such fluids (blood, CSF, urine, saliva, mammary fluid).

6. The method of claim 4, wherein said animal tissue is diseased or injured.

7. The method of claim 6, wherein said diseased tissue is cancerous, inflamed, infected, congenitally diseased or functionally compromised (diabetes, neurodegenerative, or atrophy).

8. The method of claim 6, wherein said injured tissue is traumatized or environmentally insulted.

9. The method of claim 1, wherein said tissue is plant tissue.

10. The method of claim 9, wherein said plant issue is leaf tissue, stalk tissue, stem tissue, root tissue, or seed tissue.

11. The method of claim 9, wherein said plant tissue is diseased or injured.

12. The method of claim 11, wherein said diseased tissue is infected, or congenitally diseased.

13. The method of claim 11, wherein said injured tissue is traumatized or environmentally insulted.

14. The method of claim 1, wherein said one or more physical or chemical treatments comprise solvent treatment, detergent treatment, lipase treatment, proteolysis, reactive agent treatment or labeling.

15. The method of claim 14, wherein labeling comprises treatment with an isotope dilution reagent, a labeled antibody, or an enzyme.

16. The method of claim 1, wherein analyzing comprises secondary ion mass spectrometry, laser desorption mass spectrometry or matrix-assisted laser desorption mass spectrometry, desorption electrospray or electrospray mass spectrometry.

17. The method of claim 1, wherein step (c) is performed in situ in said microregion.

18. The method of claim 1, wherein step (c) is performed after removing said protein sample from said microregion.

19. The method of claim 2, wherein step (c) is performed in situ in said spatially discrete microregions.

20. The method of claim 2, wherein said first and second spatially discrete microregions receive distinct physical or chemical treatments.

21. The method of claim 2, wherein said first and second spatially discrete microregions receive the same physical or chemical treatment.

22. The method of claim 2, wherein said first spatially discrete microregion or tissue adjacent to said first spatially microregion is subject to a first test condition prior to step (b).

23. The method of claim 22, wherein said first test condition comprises a drug treatment, a nutrient treatment, a hormone treatment, an enzyme treatment, or a cytokine treatment.

24. The method of claim 22, further subjecting said first spatially discrete microregion or tissue adjacent to a said first spatially discrete microregion to a second test condition prior to step (b).

25. The method of claim 22, wherein said second test condition is different from said first test condition.

26. The method of claim 22, wherein said second test condition is the same as said first test condition.

27. The method of claim 1, wherein said first spatially discrete microregion is subjected to at least a second physical or chemical treatment.

28. The method of claim 27, wherein said second physical or chemical treatment is distinct from said first treatment.

29. The method of claim 1, wherein said microregion is a microwell in said tissue.

30. The method of claim 29, wherein said microwell are between 5 and 200 microns.

31. The method of claim 30, wherein said microwell are between 10 and 100 microns.

32. The method of claim 31, wherein said microwell are about 50 microns.

33. The method of claim 1, wherein said microregion is a tissue cylinder or plug.

34. The method of claim 1, further comprising generating said microregion.

35. The method of claim 34, further comprising generating a plurality of microregions or a microregion array.

36. The method of claim 2, wherein said intact tissue comprises 6 microregions.

37. The method of claim 2, wherein said intact tissue comprises 24 microregions.

38. The method of claim 2, wherein said intact tissue comprises 96 microregions.

39. The method of claim 1, wherein one or both of steps (b) and (c) are automated.

40. The method of claim 2, wherein one or both of steps (d) and (e) are automated.

41. A method for analyzing nucleic acid content in a tissue comprising:

(a) providing an intact tissue comprising a first spatially discrete microregion;

(b) subjecting said microregion to one or more physical or chemical treatments; and

(c) analyzing a nucleic acid sample from said microregion, thereby providing analysis of nucleic acid content in said tissue.

42. A method for analyzing lipid content in a tissue comprising:

(a) providing an intact tissue comprising a first spatially discrete microregion;

(b) subjecting said microregion to one or more physical or chemical treatments; and

(c) analyzing a lipid sample from said microregion, thereby providing analysis of lipid content in said tissue.

43. A method for analyzing carbohydrate content in a tissue comprising:

(a) providing an intact tissue comprising a first spatially discrete microregion;

(b) subjecting said microregion to one or more physical or chemical treatments; and

(c) analyzing a carbohydrate sample from said microregion, thereby providing analysis of carbohydrate content in said tissue.

44. A method for analyzing the delivery of an exogenous agent to a tissue comprising:

(a) providing an intact tissue comprising a first spatially discrete microregion;

(b) contacting said tissue with said exogenous agent;

(c) subjecting at said first spatially discrete microregion to one or more physical or chemical treatments; and

(d) analyzing a sample from said first spatially discrete microregion, thereby providing analysis of the delivery of said exogenous agent to said tissue.

45. The method of claim 44, wherein said exogenous agent is a peptide or a polypeptide.

46. The method of claim 44, wherein said exogenous agent is a nucleic acid.

47. The method of claim 46, wherein said nucleic acid is an expression construct.

48. The method of claim 47, wherein said expression construct encodes an antisense molecule, a ribozyme, an siRNA, an enzyme, a single-chain antibody, a hormone, a toxin, a tumor suppressor, an inducer of apoptosis, a cell cycle regulator, a cytokine, or a growth factor.

49. The method of claim 44, wherein said exogenous agent is an organopharmaceutical.

50. The method of claim 44, wherein said exogenous agent is a metabolite.

51. The method of claim 44, wherein analyzing comprises secondary ion mass spectrometry, laser desorption mass spectrometry or matrix-assisted laser desorption mass spectrometry, desorption electrospray or electrospray mass spectrometry.

52. The method of claim 44, wherein said intact tissue comprises at least a second spatially discrete microregion, and said method further comprises subjecting said second spatially discrete microregion to one or more physical or chemical treatments, and analyzing a sample from said second spatially discrete microregion.

53. The method of claim 52, wherein the samples from said first and second spatially discrete microregions are compared.

54. The method of claim 44, wherein said microregion is a microwell in said tissue.

55. The method of claim 44, wherein said microregion is a tissue plug or cylinder.

56. The method of claim 55, wherein said tissue plug or cylinder is comprised with an array of tissue plugs or cylinders.

57. The method of claim 44, wherein the exogenous agent is delivered to the tissues as a whole.

58. The method of claim 44, wherein the exogenous agent is delivered to the microregion.

59. A method for analyzing an endogenous metabolite in a tissue comprising:

(a) providing an intact tissue comprising a first spatially discrete microregion;

(b) subjecting said microregion to one or more physical or chemical treatments; and

(c) analyzing a endogenous metabolite sample from said microregion, thereby providing analysis of endogenous metabolite content in said tissue.

60. The method of claim 59, wherein said endogenous metabolite is an organic acid metabolite, peptide metabolites, or a part of a sugar.