Systems and methods for automated analysis of cells and tissues

• US 20080013816A1
• Filed: 04/23/2007
• Published: 01/17/2008
• Est. Priority Date: 04/20/2001
• Status: Active Grant

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Camp, Robert, Rimm, David
• Granted Patent: US 8,036,833 B2
• Time in Patent Office: Days
• US Class Current: 382/133
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G06T 2207/20036 Morphological image processing
• CPC Class Codes: G06T 2207/20101 Interactive definition of point of interest, landmark or seed
• CPC Class Codes: G06T 2207/20221 Image fusion; Image merging
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 2207/30072 Microarray; Biochip, DNA array; Well plate
• CPC Class Codes: G06T 2207/30096 Tumor; Lesion
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/0014 using an image reference approach
• CPC Class Codes: G06T 7/41 based on statistical description of texture
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts

Abstract

Systems and methods for rapidly analyzing cell containing samples, for example to identify morphology or to localize and quantitate biomarkers are disclosed.

Claims

1-8. -8. (canceled)

11-38. -38. (canceled)

40. A computer implemented method for localizing and quantitating a particular biomarker within a first marker defined subcellular compartment relative to a second marker defined subcellular compartment present in individual cells of interest contained in a tissue sample comprising:

a) incubating the tissue sample with a first stain that specifically labels the first marker defined subcellular compartment, second stain that specifically labels the second marker defined subcellular compartment, and a third stain that specifically labels the biomarker;

b) obtaining a high resolution image of each of the first, the second, and the third stain in the tissue sample using an upright or inverted optical microscope so as to obtain;

i) a first image of the first marker defined subcellular compartment;

ii) a second image of the second marker defined subcellular compartment; and

iii) a third image of the biomarker;

 

wherein each image comprises 1024×

1024 pixel locations;

c) reiteratively analyzing each pixel location in the first and the second image so as to assign each such pixel location to the first, the second or neither subcellular compartment based upon an intensity value of the first stain relative to the second stain at that pixel location;

d) analyzing in the third image the pixel locations assigned to the first or the second subcellular compartment in step (c) so as to identify those pixel locations having an intensity value indicative of the third stain, and determining the total intensity value of the third stain at the pixel locations in each of the first and the second subcellular compartment;

so as to thereby localize and quantitate the biomarker in the first subcellular compartment relative to the second subcellular compartment.

9. The method of claim 40, wherein the subcellular compartment is selected from the group consisting of a cell nucleus, a cytoplasm, a nuclear membrane, a cellular membrane, a mitochondria, an endoplasmic reticulum, a peroxisome and a lysosome.

10. The method of claim 40, wherein the biomarker is selected from the group consisting of a protein, a peptide, a nucleic acid, a lipid or a carbohydrate.

39. The method of claim 40, wherein each of the first, the second and the third stain comprises a fluorophore.

41. The method of claim 40, wherein the quantitation of the biomarker present within the first or the second subcellular compartment comprises summing the intensity values of the third stain at the pixel locations within such subcellular compartment and dividing the sum by the number of pixels in such subcellular compartment.

42. The method of claim 40, wherein a pixel location not assigned to the first or the second subcellular compartment is assigned to a third subcellular compartment.

43. The method of claim 40, wherein the tissue has a thickness of about five microns.

44. The method of claim 40, wherein the first subcellular compartment is a cellular membrane and the second subcellular compartment is a cell nucleus.

45. The method of claim 40, wherein the tissue sample is a fixed tissue section.

46. The method of claim 40, wherein the first or the second stain reacts with a marker that is selected from

the group consisting of cytokeratin, beta catenin, alpha catenin and vimentin.

47. The method of claim 40, wherein at least one of the first, the second or the third stains comprises a fluorophore selected from the group consisting of 4′

,6-diamidino-2-phenylindole (DAPI), Cy3 and Cy-5-tyramide.

48. The method of claim 40, wherein the biomarker is selected from the group consisting of Her-2/neu, estrogen receptor, progesterone receptor and epidermal growth factor receptor.

49. The method of claim 40, further comprising after step (b) but before step (c) performing a pseudo-deconvolution step comprising:

1) obtaining an out-of-focus image of each of the first, the second and the third stain in the tissue sample wherein each image has an out-of-focus intensity value for each pixel location; and

2) subtracting the out-of-focus intensity value for each pixel location from the intensity value at such pixel location in the first, the second and the third images of step (b);

so as to thereby obtain a processed image for each stain, corrected for background.

50. The method of claim 40, wherein a mask is applied to the first, the second and the third images.