Spectral imaging

• US 20070016082A1
• Filed: 09/18/2006
• Published: 01/18/2007
• Est. Priority Date: 09/23/2003
• Status: Active Grant

• Current Assignee: Cambridge Research & Instrumentation Incorporated (Revvity, Inc.)
• Original Assignee: Cambridge Research & Instrumentation Incorporated (Revvity, Inc.)
• Inventors: Levenson, Richard, Cronin, Paul
• Granted Patent: US 8,391,961 B2
• Time in Patent Office: Days
• US Class Current: 600/476
• CPC Class Codes: A61B 5/0059 using light, e.g. diagnosis by transillumination, diascopy, fluorescence A61B5/0093, A61B5/1455, A61B5/02416 and A61B5/0261 take precedence
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01J 3/32 Investigating bands of a spectrum in sequence by a single detector
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01J 3/453 by correlation of the amplitudes
• CPC Class Codes: G01N 21/255 Details, e.g. use of specially adapted sources, lighting or optical systems
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/125 Digital circuitry
• CPC Class Codes: G01N 33/4833 of solid biological material, e.g. tissue samples, cell cultures tissue in vivo A61B5/00; cell suspensions G01N33/48735

Abstract

Apparatus and methods are provided for the imaging of structures in deep tissue within biological specimens, using spectral imaging to provide highly sensitive detection. By acquiring data that provides a plurality of images of the sample with different spectral weightings, and subsequent spectral analysis, light emission from a target compound is separated from autofluorescence in the sample. With the autofluorescence reduced or eliminated, an improved measurement of the target compound is obtained.

Claims

1-40. -40. (canceled)

41. A method comprising:

providing spectrally resolved information about light coming from different spatial locations of a biological sample in response to an illumination of the sample, wherein the light includes contributions from different components in the sample;

decomposing at least some of the spectrally resolved information for each of at least some of the different spatial locations into a contribution from a spectral estimate of a pure spectrum for at least a first one of the components in the sample; and

constructing an image of the sample based on the decomposition to preferentially show a selected one of the components,

wherein the estimate of the pure spectrum for the first component is derived from at least part of the spectrally resolved information corresponding to a first set of one or more of the different spatial locations and a spectral estimate of a pure spectrum for a second one of the components.

42. The method of claim 41, wherein the sample comprises deep tissue, tissue slices, cells, subdermal tissue, or a microscope slide carrying biological material.

43. The method of claim 41, wherein the decomposing comprises decomposing the spectrally resolved information for each of at least some of the different spatial locations into contributions from estimates of pure spectra for at least some of the components in the sample.

44. The method of claim 41, wherein the decomposition is a linear decomposition.

45. The method of claim 44, wherein the decomposition comprises solving at least one component of a matrix equation in which one matrix is based on the spectrally resolved information and another matrix is based on the estimate of the pure spectrum for the first component and an estimate for the pure spectrum of at least one additional component.

46. The method of claim 41, wherein the pure spectrum for the first component corresponds to the spectrally resolved information that would result if only the first component contributes to the light.

47. The method of claim 41, wherein the spectral estimate of the pure spectrum for the second component is provided independently of the spectrally resolved information.

48. The method of claim 41, wherein the spectral estimate of the pure spectrum for the second component is determined from the spectrally resolved information.

49. The method of claim 48, wherein the spectral estimate of the pure spectrum for the second component is determined from the spectrally resolved information by associating a region of one or more of the spatial locations with the second component.

50. The method of claim 41, wherein the spatial locations in the first set are spatial location in which the light includes contributions from multiple ones of the components.

51. The method of claim 41, wherein deriving the spectral estimate of the pure spectrum for the first component comprises calculating a remainder spectrum based on the spectrally resolved information from the first set and the spectral estimate for the second component.

52. The method of claim 51, wherein the remainder spectrum is calculated at each of one or more of the spatial locations in the first set of spatial locations.

53. The method of claim 41, wherein deriving the spectral estimate of the pure spectrum for the first component comprises adjusting values corresponding to the spectrally resolved information for the first set of spatial locations to remove a contribution from the second component based on the spectral estimate for the second component.

54. The method of claim 53, wherein the removed contribution is a maximal contribution.

55. The method of claim 41, wherein the spectrally resolved information comprises information about a set of images in which the light coming from the sample is spectrally filtered, wherein the spectral filtering for each image corresponds to a different spectral weighting function.

56. The method of claims 55, wherein the different spatial locations correspond to common pixels in the set of images.

57. The method of claims 55, wherein the different spectral weighting functions correspond to different spectral bands.

58. The method of claims 55, wherein the set of images comprises four or more images.

59. The method of claim 56, wherein the information about the set of images comprises a series of values at each of the pixels, wherein each value is related to an intensity of the light coming from the sample with respect to a corresponding one of the spectral weighting functions.

60. The method of claim 41, wherein the spectrally resolved information for each spatial location comprises information corresponding to at least four different spectral weighting functions.

61. The method of claim 41, wherein the spectrally resolved information comprises a spectral image cube.

62. The method of claim 41, wherein the light coming from the sample comprises fluorescence from the sample.

63. The method of claim 41, wherein at least one of the components relates to auto fluorescence.

64. The method of claim 41, wherein at least one of the components comprises a target compound.

65. The method of claim 64, wherein the selected component is the component comprising the target compound.

66. The method of claim 64, wherein the target compound comprises a fluorescent protein or a quantum dot.

67. The method of claim 41, further comprising illuminating the sample and collecting the spectrally resolved information.

68. The method of claim 41, wherein collecting the spectrally resolved information comprises using a liquid crystal tunable spectral filter, an acousto-optical tunable spectral filter, a set of spectral filters, or a spectral filter wheel.

69. The method of claim 41, wherein the image of the selected component comprises an image in which signal from the other components is reduced relative to signal from the selected component.

70. The method of claim 41, further comprising constructing a second image of the sample based on the decomposition to preferentially show a second one of the components.

76. Apparatus comprising a computer-readable medium storing a program that causes a processor to carry out the steps of claims 41.

71. An apparatus comprising:

a sample holder configured to support a deep tissue sample;

an illumination source to illuminate the sample;

a detector positioned to detect light from the sample; and

an electronic processor coupled to the detector, wherein the electronic processor is configured to;

(i) provide spectrally resolved information about light coming from different spatial locations of a sample, wherein the light includes contributions from different components in the sample;

(ii) decompose at least some of the spectrally resolved information for each of at least some of the different spatial locations into contributions from spectral estimates associated with at least some of the components in the sample; and

(iii) construct a deep tissue image of the sample based on the decomposition to preferentially show a selected one of the components.

74. The apparatus of claims 71, 72, or 73, further comprising a spectral filtering means positioned between the sample and the detector.

75. The apparatus of claim 74, wherein the spectral filtering means comprises a liquid crystal tunable spectral filter, an acousto-optical tunable spectral filter, a set of spectral filters, or a spectral filter wheel.

72. An apparatus comprising:

a sample holder configured to support a deep tissue sample;

an illumination source to illuminate the sample;

a detector positioned to detect light from the sample; and

an electronic processor coupled to the detector, wherein the electronic processor is configured to;

(i) provide spectrally resolved information about light coming from different spatial locations of a sample, wherein the light includes contributions from different components in the sample and the spectrally resolved information for each spatial location comprises information corresponding to at least four different spectral weighting functions; and

(ii) construct a deep tissue image of the sample based on the spectrally resolved information to preferentially show a selected one of the components.

73. An apparatus comprising:

a sample holder configured to support a sample;

an illumination source to illuminate the sample;

a detector positioned to detect light from the sample; and

an electronic processor coupled to the detector, wherein the electronic processor and is configured to;

(i) provide spectrally resolved information about light coming from different spatial locations of a sample, wherein the light includes contributions from different components in the sample;

(ii) decompose at least some of the spectrally resolved information for each of at least some of the different spatial locations into a contribution from a spectral estimate of a pure spectrum for at least a first one of the components in the sample; and

(iii) construct an image of the sample based on the decomposition to preferentially show a selected one of the components, wherein the estimate of the pure spectrum for the first component is derived from at least part of the spectrally resolved information corresponding to a first set of one or more of the different spatial locations and a spectral estimate of a pure spectrum for a second one of the components.

77. A method comprising:

providing a set of images of spectrally filtered radiation emitted from a biological sample in response to an illumination, wherein the sample comprises a component supporting a target compound, the emitted radiation comprises emission from the target compound and emission from one or more additional components in the sample, and each image corresponds to a different spectral weighting function for a common set of pixels; and

processing the images of the spectrally filtered radiation to construct an output image of the sample in which signal from the additional components is reduced relative to signal from the target compound,

wherein the processing comprises calculating a remainder spectrum for one or more pixels in the set of images based on an estimate for an emission spectrum of at least one of the components.