Title:
Spectral volume microprobe arrays
Document Type and Number:
Kind Code:
A1

Abstract:
Methods and apparatus are provided for determining a characteristic of a sample of a material by the interaction of electromagnetic radiation with the sample. The apparatus includes a source of electromagnetic radiation, an optical assembly and a detector. The optical assembly sequentially illuminates a plurality of volume elements in the sample with an intensity distribution in the sample that drops off substantially monotonically from a first region in a first optical path and collects electromagnetic radiation emanating from each of the volume elements. The optical assembly collects the electromagnetic radiation emanating from each of the volume elements with a collected distribution that drops off substantially monotonically from a second region in a second optical path. The first and second regions at least partially overlap in each of the volume elements. The detector detects the collected electromagnetic radiation emanating from each of the sequentially illuminated volume elements to produce responses representative of the characteristic in each of the volume elements.
Representative Image:
Inventors:
Modell, Mark (Brookline, MA, US)
Hed, Ze?apos Ev A. (Nashua, NH, US)
Application Number:
09/841325
Publication Date:
11/15/2001
Filing Date:
04/24/2001
View Patent Images:
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Primary Class:
International Classes:
(IPC1-7): A61B005/00; A61B006/00
Attorney, Agent or Firm:
TESTA, HURWITZ & THIBEAULT, LLP (HIGH STREET TOWER, BOSTON, MA, 02110, US)
Claims:

What is claimed is:



1. Apparatus for determining a characteristic of a sample of material by the interaction of electromagnetic radiation with the sample, comprising: a source of electromagnetic radiation; illuminating optics for sequentially illuminating a plurality of volume elements in the sample with an intensity distribution in the sample that drops off substantially monotonically from a first region in a first optical path; collecting optics for collecting electromagnetic radiation emanating from each of said volume elements, said collecting optics collecting said electromagnetic radiation emanating from each of said volume elements with a collection distribution that drops off substantially monotonically from a second region in a second optical path, said first and second regions at least partially overlapping in each of said volume elements, said illuminating and collecting optics each having respective field stops whose dimensions are large compared to a quotient of wavelength of said electromagnetic radiation divided by a working numerical aperture of said illuminating and collecting optics, respectively, measured from said respective field stops; and a detector for detecting the collected electromagnetic radiation emanating from each of said sequentially illuminated volume elements to produce a response representative of said characteristic in each of said volume elements, wherein the sample of material comprises a biological tissue.

2. Apparatus as defined in claim 1 wherein at least one of the illuminating and the collecting optics comprises means for determining said characteristic from a three dimensional array of said volume elements.

3. Apparatus as defined in claim 1 wherein the field stops of said illuminating optics comprises an array of individually controllable illuminating optical shutters for sequentially illuminating said volume elements.

4. Apparatus as defined in claim 3 wherein the field stops of said collecting optics comprises an array of individually controllable collection optical shutters for sequentially collecting electromagnetic radiation emanating from each of said volume elements.

5. Apparatus as defined in claim 1 wherein said illuminating optics comprises an array of individually controllable illuminating elements for sequentially illuminating said volume elements and said collecting optics comprises an array of individually controllable collection elements for sequentially collecting electromagnetic radiation emanating from each of said volume elements.

6. Apparatus for determining a characteristic of a sample of material by the interaction of electromagnetic radiation with the sample, comprising: a source of electromagnetic radiation; an optical assembly for sequentially illuminating a plurality of volume elements in the sample with an intensity distribution in the sample that drops off substantially monotonically from a first region in a first optical path and for collecting electromagnetic radiation emanating from each of said volume elements, said optical assembly collecting said electromagnetic radiation emanating from each of said volume elements with a collection distribution that drops off substantially monotonically from a second region in a second optical path, said first and second regions at least partially overlapping in each of said volume elements, said optical assembly comprising at least one array of field stops whose dimensions are large compared to a quotient of wavelength of said electromagnetic radiation divided by a working numerical aperture of said optical assembly, measured from said field stops; and a detector for detecting the collected electromagnetic radiation emanating from each of said sequentially illuminated volume elements to produce responses representative of said characteristic in each of said volume elements, wherein said sample comprises a biological tissue.

7. Apparatus as defined in claim 6 wherein said array of field stops comprises a single array of individually controllable optical shutters for sequentially illuminating said volume elements and for sequentially collecting electromagnetic radiation emanating from each of said volume elements, wherein said first and second optical paths are the same.

8. Apparatus as defined in claim 7 wherein said array of optical shutters comprises an array of individually controllable liquid crystal shutter elements.

9. Apparatus as defined in claim 7 wherein said array of optical shutters comprises an array of individually controllable polymer dispersed liquid crystal shutter elements.

10. Apparatus as defined in claim 7 wherein said array of optical shutters comprises an array of individually controllable ferroelectric shutter elements.

11. Apparatus as defined in claim 7 wherein said array of optical shutters comprises an array of individually controllable piezoelectric bimorph shutter elements.

12. Apparatus as defined in claim 7 wherein said array of optical shutters comprises a micromachined array of individually controllable, electrostatically movable shutter elements.

13. Apparatus as defined in claim 6 further including means for moving at least a portion of said optical assembly with respect to the sample so as to vary the locations of said volume elements within the sample along the optical axis of said first and second optical paths.

14. Apparatus as defined in claim 7 wherein said optical assembly further includes an optical objective between said array of optical shutters and the sample.

15. Apparatus as defined in claim 14 wherein said optical objective comprises a single objective lens aligned with said array of optical shutters.

16. Apparatus as defined in claim 14 wherein said optical objective comprises an array of microlens elements respectively aligned with said optical shutters.

17. Apparatus as defined in claim 7 wherein said array of optical shutters is subdivided into noninterfering zone arrays and electromagnetic radiation is collected simultaneously from said noninterfering zone arrays.

18. Apparatus as defined in claim 7 wherein said array of optical shutters comprises a plurality of rows and columns of shutter elements.

19. Apparatus as defined in claim 7 wherein said array of optical shutters comprises an array of radially distributed shutter elements.

20. Apparatus as defined in claim 7 wherein said array of optical shutters comprises a linear array of shutter elements.

21. Apparatus as defined in claim 7 wherein said array of optical shutters comprises a circumferential array of shutter elements.

22. Apparatus as defined in claim 6 wherein said array of field stops comprises an array of individually movable micromirrors, each of said micromirrors being movable between an active position for directing illumination from said source to the sample and for directing collected electromagnetic radiation from the sample to said detector, and an inactive position.

23. Apparatus as defined in claim 6 wherein said array of field stops comprises an array of individually movable micromirrors, each of said micromirrors comprising an off axis segment of a paraboloid of revolution, said micromirrors being movable in pairs between active positions and inactive positions, one micromirror of an active pair directing illumination from said source to the sample and the other micromirror of the active pair directing electromagnetic radiation emanating from the sample to said detector.

24. Apparatus as defined in claim 23 wherein micromirrors of a first group of said micromirrors sequentially direct illumination from said source to the sample and micromirrors of a second group of said micromirrors sequentially direct collected electromagnetic radiation from the sample to said detector.

25. Apparatus as defined in claim 23 wherein said array of field stops further comprises means for moving each of said micromirrors between an illumination position for directing illumination from said source to the sample and a collection position for directing collected electromagnetic radiation from the sample to said detector, wherein each of said micromirrors is used for illumination and collection at different times.

26. Apparatus as defined in claim 6 wherein said optical assembly comprises a bundle of optical fibers and an optical fiber switching device for sequentially activating each of said optical fibers for directing illumination from said source to the sample and for directing collected electromagnetic radiation from the sample to said detector.

27. Apparatus as defined in claim 6 wherein said optical assembly comprises a bundle of optical fibers and an array of optical shutters positioned at one end of said bundle of optical fibers so that said optical shutters are respectively aligned with said optical fibers, said array of optical shutters sequentially illuminating said volume elements and sequentially collecting electromagnetic radiation emanating from each of said volume elements.

28. Apparatus as defined in claim 6 wherein said detector comprises a single optical detector for detecting collected electromagnetic radiation from each of said volume elements.

29. Apparatus as defined in claim 7 wherein said detector comprises a plurality of detector elements corresponding to said optical shutters or to groups of said optical shutters.

30. Apparatus as defined in claim 6 wherein said source comprises a laser source.

31. Apparatus as defined in claim 6 wherein said source comprises a nitrogen laser.

32. Apparatus as defined in claim 6 wherein said source comprises a broad spectral band light source.

33. Apparatus as defined in claim 32 wherein said source comprises a xenon discharge lamp.

34. Apparatus as defined in claim 32 wherein said source comprises a halogen incandescent lamp.

35. Apparatus as defined in claim 6 wherein said source comprises an ultraviolet wavelength source.

36. Apparatus as defined in claim 6 wherein said optical assembly includes an optical filter for tailoring the spectrum of the illumination.

37. Apparatus as defined in claim 6 wherein said source comprises a plurality of light sources and means for activating said light sources at different times.

38. Apparatus as defined in claim 6 wherein said optical assembly further includes means for modulating the illumination of the sample.

39. Apparatus as defined in claim 6 wherein said response comprises natural fluorescence of tissue after illumination by a narrow wavelength excitation.

40. Apparatus as defined in claim 6 wherein said response is produced by selectively absorbed dye in pathological tissue.

41. Apparatus as defined in claim 6 wherein said detector comprises means for detecting responses produced by Raman scattering.

42. Apparatus as defined in claim 6 wherein said detector comprises means for detecting a combination of backscattering and reflection from said volume elements.

43. Apparatus as defined in claim 6 wherein said at least one array of field stops comprises an array of individually controllable illuminating optical shutters for sequentially illuminating said volume elements and an array of individually controllable collection optical shutters for sequentially collecting electromagnetic radiation emanating from each of said volume elements, and wherein said optical assembly further comprises illuminating light conditioning optics for directing illumination from said source to said array of illuminating optical shutters, an illuminating objective for focusing illumination on said volume elements, a collection objective for focusing said collection optical shutters on said volume elements and collection light conditioning optics for directing said collected electromagnetic radiation from said array of collection optical shutters to said detector.

44. Apparatus as defined in claim 6 wherein said at least one array of field stops comprises an array of individually controllable illuminating optical shutters for sequentially illuminating said plurality of volume elements and an array of individually controllable collection optical shutters for sequentially collecting electromagnetic radiation emanating from each of said volume elements.

45. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises an array of individually controllable liquid crystal shutter elements.

46. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises an array of individually controllable polymer dispersed liquid crystal shutter elements.

47. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises an array of individually controllable ferroelectric shutter elements.

48. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises an array of individually controllable piezoelectric bimorph shutter elements.

49. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises a micromachined array of individually controllable, electrostatically movable shutter elements.

50. Apparatus as defined in claim 44 wherein said optical assembly further includes a single illuminating objective lens between said array of illuminating optical shutters and the sample, and a single collection objective lens between the sample and said array of collection optical shutters.

51. Apparatus as defined in claim 44 wherein said optical assembly further includes an array of illuminating objective microlens elements respectively aligned with the optical shutters of said array of illuminating optical shutters and positioned between said array of illuminating optical shutters and the sample, and an array of collection objective microlens elements respectively aligned with the optical shutters of said array of collection optical shutters and positioned between the sample and said array of collection optical shutters.

52. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters are subdivided into noninterfering zone arrays and electromagnetic radiation is collected simultaneously from said noninterfering zone arrays.

53. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises a plurality of rows and columns of shutter elements.

54. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises an array of radially distributed shutter elements.

55. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises a linear array of shutter elements.

56. Apparatus as defined in claim 44 wherein said array of illuminating optical shutters and said array of collection optical shutters each comprises a circumferential array of shutter elements.

57. Apparatus as defined in claim 6 wherein said optical assembly comprises illuminating optics for sequentially illuminating said plurality of volume elements and collecting optics for collecting electromagnetic radiation emanating from each of said volume elements, said illuminating optics comprising an illuminating bundle of optical fibers and illuminating control means for sequentially activating each of said optical fibers for directing illumination from said source to the sample, said collecting optics comprising a collection bundle of optical fibers and collection control means for sequentially activating each of said optical fibers in said collection bundle for directing collected electromagnetic radiation from the sample to said detector.

58. Apparatus as defined in claim 57 wherein said illuminating control means comprises a first rotating mirror for sequentially directing illumination into the optical fibers of said illumination bundle and wherein said collection control means comprises a second rotating mirror for sequentially directing collected electromagnetic radiation from the optical fibers of said collection bundle to said detector.

59. Apparatus as defined in claim 57 wherein said illuminating control means comprises an array of individually controllable illuminating optical shutters respectively aligned with the optical fibers of said illuminating bundle and wherein said collection control means comprises an array of individually controllable collection optical shutters respectively aligned with the optical fibers of said collection bundle.

60. Apparatus as defined in claim 6 further comprising a spectral analyzer disposed between said optical assembly and said detector.

61. Apparatus as defined in claim 6 further comprising a temporal interferometer disposed between said optical assembly and said detector.

62. Apparatus as defined in claim 6 further comprising a spatial interferometer disposed between said optical assembly and said detector.

63. Apparatus as defined in claim 6 further comprising a Hadamard encodement mask disposed between said optical assembly and said detector.

64. A method for determining a characteristic of a sample of material by the interaction of electromagnetic radiation with the sample, comprising the steps of: providing an optical assembly adapted for positioning in proximity to the sample, wherein the sample comprises a biological tissue, sequentially illuminating, with an optical assembly, a plurality of volume elements in the sample by directing electromagnetic radiation into the sample with an intensity distribution in the sample that drops off substantially monotonically from a first region in a first optical path; sequentially collecting, with said optical assembly, electromagnetic radiation emanating from each of said sequentially illuminated volume elements with a collection distribution that drops off substantially monotonically from a second region in a second optical path, said first and second regions at least partially overlapping in each of said volume elements, said optical assembly comprising at least one array of field stops whose dimensions are large compared to a quotient of wavelength of said electromagnetic radiation divided by a working numerical aperture of said optical assembly, measured from said field stops; and detecting the collected electromagnetic radiation emanating from each of said sequentially illuminated volume elements to produce a response representative of said characteristic in each of said volume elements.

65. A method as defined in claim 64 wherein the steps of sequentially illuminating and sequentially collecting are performed with said optical assembly, wherein said at least one array of field stops comprises a single array of individually controllable optical shutters.

66. A method as defined in claim 64 wherein said method further includes the step of moving at least a portion of said optical assembly with respect to the sample so as to vary the locations of said volume elements within the sample along the optical axis of said first and second optical paths.

67. A method as defined in claim 64 wherein the step of illuminating a plurality of volume elements includes simultaneously illuminating two or more noninterfering volume elements and wherein the step of collecting electromagnetic radiation includes simultaneously collecting electromagnetic radiation emanating from said noninterfering volume elements.

68. A method as defined in claim 64 wherein said array of field stops comprises an array of individually movable micromirrors, said method further comprising sequentially moving the micromirrors of said array between an active position for directing illumination to the sample and for directing collected electromagnetic radiation from the sample to a detector, and an inactive position.

69. A method as defined in claim 64 wherein said array of field stops comprises an array of individually movable micromirrors, each of said micromirrors comprising an off axis segment of a paraboloid of revolution, said method further comprising moving pairs of said micromirrors between active positions and inactive positions, one micromirror of an active pair directing illumination to the sample and the other micromirror of the active pair directing electromagnetic radiation emanating from the sample to a detector.

70. A method as defined in claim 64 wherein the step of illuminating a plurality of volume elements includes directing illumination through a bundle of illuminating optical fibers.

71. A method as defined in claim 64 wherein the step of collecting electromagnetic radiation includes directing collected electromagnetic radiation through a bundle of collection optical fibers.

72. A method as defined in claim 64 wherein the step of illuminating a plurality of volume elements includes modulating the illumination of the sample.

73. A method as defined in claim 64 wherein the steps of sequentially illuminating and sequentially collecting are performed with said optical assembly, wherein said at least one array of field stops comprises an array of individually controllable illuminating optical shutters and an array of individually controllable collection optical shutters.

74. A method as defined in claim 64 wherein the steps of sequentially illuminating and sequentially collecting are performed with said optical assembly, wherein said at least one array of field stops comprises an array of individually controllable illuminating elements and an array of individually controllable collection elements.

75. A method as defined in claim 64 wherein the step of illuminating a plurality of volume elements includes illuminating said volume elements at different times with sources having different spectra.

76. A method as defined in claim 64 wherein the step of detecting the collected electromagnetic radiation is performed by an array of detector elements.

77. Apparatus as defined in claim 1 wherein the illuminating optics, the collecting optics and the detector include means for producing a response representative of a characteristic of biological tissue.

78. Apparatus as defined in claim 1 wherein said source, said illuminating optics, said collecting optics and said detector are configured for determining said characteristic in vitro.

79. Apparatus as defined in claim 1 wherein said source, said illuminating optics, said collecting optics and said detector are configured for determining said characteristic in vivo.

80. Apparatus as defined in claim 1 wherein said detector includes means for producing a response representative of at least one pathology.

81. Apparatus as defined in claim 1 wherein said detector includes means for producing a response representative of cancer.

82. Apparatus as defined in claim 80 further including means for presenting the response as an image of the spatial distribution of said at least one pathology.

83. A method as defined in claim 64 wherein the steps of sequentially illuminating, sequentially collecting and detecting are carried out with biological tissue as the sample.

84. A method as defined in claim 64 wherein the steps of sequentially illuminating, sequentially collecting and detecting are carried out in vitro.

85. A method as defined in claim 64 wherein the steps of sequentially illuminating, sequentially collecting and detecting are carried out in vivo.

86. A method as defined in claim 64 wherein the step of detecting includes producing a response representative of at least one pathology.

87. A method as defined in claim 64 wherein the step of detecting includes producing a response representative of cancer.

88. A method as defined in claim 86 firther including the step of presenting the response as an image of the spatial distribution of at least one pathology.

89. Apparatus as defined in claim 1 wherein said biological tissue comprises an in vivo biological tissue.

90. Apparatus as defined in claim 88 further comprising a housing adapted for introducing said illuminating optics and said collecting optics into proximity to said in vivo biological tissue.

91. Apparatus as defined in claim 89 wherein said housing is adapted for permitting only a single intervention whereby said illuminating optics and said collecting optics are introduced into proximity to said in vivo biological tissue.

92. Apparatus as defined in claim 90 wherein said housing is adapted for being disposed of after use.

93. Apparatus as defined in claim 6 wherein said biological tissue comprises an in vivo biological tissue.

94. Apparatus as defined in claim 92 further comprising a housing adapted for introducing said optical assembly into proximity to said in vivo biological tissue.

95. Apparatus as defined in claim 93 wherein said housing is adapted for permitting only a single intervention whereby the optical assembly is introduced into proximity to said in vivo biological tissue.

96. Apparatus as defined in claim 94 wherein said housing is adapted for being disposed of after use.

97. Apparatus for covering an optical assembly, comprising: a sheath positioned external to a surface of the optical assembly, whereby the sheath is interposed between the surface of the optical assembly and a biological tissue; and a region in the sheath adapted for the transmission of optical signals between the optical assembly and the biological tissue.

98. Apparatus as defined in claim 96 wherein the region in the sheath is configured as a window.

99. Apparatus as defined in claim 97 wherein the window is in contact with the optical assembly.

100. Apparatus as defined in claim 96 wherein the sheath comprises a region shaped as a cylinder.

101. Apparatus as defined in claim 96 wherein the sheath is adapted for a single use.

102. Apparatus as defined in claim 96 further comprising a fastening mechanism whereby the sheath can be attached to the optical assembly.

103. Apparatus as defined in claim 101 wherein the fastening mechanism is adapted for preventing the sheath from being used with the optical assembly on more than one occasion.

104. A method for diagnosing a biological tissue, comprising: providing an optical assembly dimensionally adapted for positioning in proximity to a biological tissue; providing a sheath adapted for covering a surface of the optical assembly, wherein the sheath is capable of transmitting optical signals between the optical assembly and the biological tissue; positioning the sheath around the optical assembly so that the sheath is interposed between the optical assembly and the biological tissue; placing the optical assembly in juxtaposition to the biological tissue for a diagnosis thereof; sequentially illuminating, with an optical assembly, a plurality of volume elements in the biological tissue by directing electromagnetic radiation into the biological tissue with an intensity distribution in the biological tissue that drops off substantially monotonically from a first region in a first optical path; sequentially collecting, with said optical assembly, electromagnetic radiation emanating from each of said sequentially illuminated volume elements with a collection distribution that drops off substantially monotonically from a second region in a second optical path, said first and second regions at least partially overlapping in each of said volume elements, said optical assembly comprising at least one array of field stops whose dimensions are large compared to a quotient of wavelength of said electromagnetic radiation divided by a working numerical aperture of said optical assembly, measured from said field stops; and detecting the collected electromagnetic radiation emanating from each of said sequentially illuminated volume elements to produce a response representative of said characteristic in each of said volume elements.

105. A method according to claim 103 further comprising attaching the sheath to the optical assembly with a fastening mechanism.

Description:

PRIOR APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application 60/115373, Jan. 11, 1999, and is a continuation-in-part of U.S. patent application Ser No. 08/782,936, filed Jan. 13, 1997.

FIELD OF THE INVENTION

[0002] The present invention provides apparatus and methods to derive spatially differentiated analytical information from an exposed surface by analyzing the results of the interaction of electromagnetic radiation with discrete volume elements of the sample. This is achieved by spatially limiting the probing beam to a small volume element and limiting the accepted response detected from the same volume element only, scanning the sample at various depths along the axis of the optical assembly formed by the beam to determine the interaction from volume elements at the different depths and collecting such data from a plurality of points in a plane generally perpendicular to the probing beam.

BACKGROUND OF THE INVENTION

[0003] An important requirement exists for an instrument that will provide rapid and automatic diagnostic information, for example of cancerous and otherwise diseased tissue. In particular, there is a need for an instrument that would map the extent and stage of cancerous tissue without having to excise a large number of tissue samples for subsequent biopsies. In the current art, the medical profession relies generally on visual analysis and biopsies to determine specific pathologies and abnormalities. Various forms of biochemical imaging are used as well. Unique optical responses of various pathologies are being exploited in attempts to characterize biological tissue as well.

[0004] These prior art techniques, however, contain serious drawback as documented in copending application Ser. Nos. 08/510,041 filed Aug. 1, 1995 and 08/510,043 filed Aug. 1, 1995, which are incorporated herein by reference.

[0005] For example, performing a tissue biopsy and analyzing the extracted tissue in the laboratory requires a great deal of time. In addition, tissue biopsies can only characterize the tissue based upon representative samples taken from the tissue. This results in a large number of resections being routinely performed to gather a selection of tissue capable of accurately representing the sample. In addition, tissue biopsies are subject to sampling and interpretation errors. Magnetic resonance imaging is a successful tool, but is expensive and has serious limitations in detecting pathologies that are very thin or in their early stages of development.

[0006] One technique used in the medical field for tissue analysis is induced fluorescence. Laser induced fluorescence utilizes a laser tuned to a particular wavelength to excite tissue and to cause the tissue to fluoresce at a set of secondary wavelengths that can then be analyzed to infer characteristics of the tissue. Fluorescence can originate either from molecules normally found within the tissue, or from molecules that have been introduced into the body to serve as marker molecules.

[0007] Although the mechanisms involved in the fluorescence response of biological tissue to UV excitation have not been clearly defined, the fluorescence signature of neoplasia appears to reflect both biochemical and morphological changes. The observed changes in the spectra are similar for many cancers, which suggest similar mechanisms are at work. For example, useful auto-fluorescence spectral markers may reflect biochemical changes in the mitochondria, e.g., in the relative concentration of nicotinamide adenine dinucleotide (NADH) and flavins. Mucosal thickening and changes in capillary profusion are structural effects that have been interpreted as causing some typical changes in the spectroscopic record.

[0008] The major molecules in biological tissue which contribute to fluorescence emission under 337 nm near UV light excitation, have been identified as tryptophan (390 nm emission), chromophores in elastin (410 nn) and collagen (300 nm), NADH (470 nm), flavins (520 nm) and melanin (540 nm). However, it should be noted that in tissue, there is some peak shifting and changes in the overall shape relative to the pure compounds. Accordingly, the sample can be illuminated with a UV beam of sufficiently short wavelength and record responses from the above enumerated wavelengths of light in order to determine the presence of each of above identified contributions to tissues types.

[0009] It has been further shown that hemoglobin has an absorption peak between 400 and 540 nm, while both oxyhemoglobin and hemoglobin have strong light absorption above 600 nm. Blood distribution may also influence the observed emission spectra of elastin, collagen, NAD, and NADH. Further compounds present in tissue which may absorb emitted light and change the shape of the emitted spectra include myoglobin, porphyrins, and dinucleotide co-enzymes.

[0010] A general belief is that neoplasia has high levels of NADH because its metabolic pathway is primarily anaerobic. The inability of cells to elevate their NAD+:NADH ratio at confluence is a characteristic of transformed cells related to their defective growth control. The ratio of NADH+:NADH is an indicator of the metabolic capability of the cell, for example, its capacity for glycolysis versus gluconeogenesis. Surface fluorescence has been used to measure the relative level of NADH in both in vitro and in vivo tissues. Emission spectra obtained from individual myocyte produces residual green fluorescence, probably originating from mitochondrial oxidized flavin proteins, and blue fluorescence is consistent with NADH of a mitochondrial origin.

[0011] Collagen, NADH, and flavin adenine dinucleotide are thought to be the major fluorophores in colonic tissue and were used to spectrally decompose the fluorescence spectra. Residuals between the fits and the data resemble the absorption spectra of a mix of oxy-and deoxy-hemoglobin; thus the residuals can be attributed to the presence of blood.

[0012] Alfano, U.S. Pat. No. 4,930,516, teaches the use of luminescence to distinguish cancerous from normal tissue when the shape of the visible luminescence spectra from the normal and cancerous tissue are substantially different, and in particular when the cancerous tissue exhibits a shift to the blue with different intensity peaks. For example, Alfano discloses that a distinction between a known healthy tissue and a suspect tissue can be made by comparing the spectra of the suspect tissue with the healthy tissue. According to Alfano, the spectra of the tissue can be generated by exciting the tissue with substantially monochromatic radiation and comparing the fluorescence induced at least at two wavelengths.

[0013] Alfano, in U.S. Pat. No. 5,042,494, teaches a technique for distinguishing cancer from normal tissue by identifying how the shape of the visible luminescence spectra from the normal and cancerous tissue are substantially different.

[0014] Alfano further teaches, in U.S. Pat. No. 5,131,398, the use of luminescence to distinguish cancer from normal or benign tissue by employing (a) monochromatic or substantially monochromatic excitation wavelengths below about 315 nm, and, in particular, between about 260 and 315 nm, and, specifically, at 300 nm, and (b) comparing the resulting luminescence at two wavelengths about 340 and 440 nm.

[0015] Alfano, however, fails to teach a method capable of distinguishing between normal, malignant, benign, tumorous, dysplastic, hyperplastic, inflamed, or infected tissue. Failure to define these subtle distinctions in diagnosis makes appropriate treatment choices nearly impossible. While the simple ratio, difference and comparison analysis of Alfano and others have proven to be useful tools in cancer research and provocative indicators of tissue status, these have not, to date, enabled a method nor provided means which are sufficiently accurate and robust to be clinically acceptable for cancer diagnosis.

[0016] It is quite evident from the above that the actual spectra obtained from biological tissues are extremely complex and thus difficult to resolve by standard peak matching programs, spectral deconvolution or comparative spectral analysis. Furthermore, spectral shifting further complicates such attempts at spectral analysis. Last, laser fluorescence and other optical responses from tissues typically fail to achieve depth resolution because either the optical or the electronic instrumentation commonly used for these techniques entail integrating the signal emitted by the excited tissue over the entire illuminated tissue volume.

[0017] Rosenthal, U.S. Pat. No. 4,017,192, describes a technique for automatic detection of abnormalities, including cancer, in multi-cellular bulk bio-medical specimens, which overcome the problems associated with complex spectral responses of biological tissues. Rosenthal teaches the determination of optical responses (transmission or reflection) data from biological tissue over a large number of wavelengths for numerous samples and then the correlation of these optical responses to conventional, clinical results to select test wavelengths and a series of constants to form a correlation equation. The correlation equation is then used in conjunction with optical responses at the selected wavelengths taken on an uncharacterized tissue to predict the status of this tissue. However, to obtain good and solid correlations, Rosenthal excises the tissues and obtains in essence a homogeneous sample in which the optical responses do not include the optical signatures of underlying tissues. Rosenthal's methods, therefore, cannot be used in vivo applications as contemplated in the present invention.

[0018] In studies carried out at the Wellman Laboratories of Photomedicine, using a single fiber depth integrating probe, Schomacker has shown that the auto-fluorescence of the signature of human colon polyps in vivo is an indicator of normality, benign hyperplasia, pre-cancerous, and malignant neoplasia. See Schomacker et al., Lasers Surgery and Medicine, 12, 63-78 (1992), and Gastroenterology 102, 1155-1160 (1992). Schomacker further teaches using multi-variant linear regression analysis of the data to distinguish neoplastic from non-neoplastic polyps. However, using Schomacker's techniques, the observation of mucosal abnormalities was hindered by the signal from the submucosa, since 87% of the fluorescence observed in normal colonic tissue can be attributed to submucosal collagen.

[0019] Accordingly, there is a need for a more effective and accurate device to characterize specimen, and particularly in vivo specimen which will obtain responses from well defined volume elements within said specimen, and present data automatically from a relatively large area comprising a plurality of such volume elements. Furthermore, there is a need for methods to automatically interpret such data in terms of simple diagnostic information on said volume elements.

[0020] In the aforementioned applications, Ser. Nos. 08/510,041 and 08/510,043, Modell, DeBaryshe and Hed taught the general principles of obtaining valuable analytical data from a volume element in a target sample by using spatial filters with dimensions that are generally larger than the diffraction limits for the wavelengths of the probing radiation. Such spatial filtration is obtained by an optical device including an illumination and a detection system both containing field stops and the field stops being conjugated to each other via the volume element to be analyzed, providing in essence a non imaging volume microprobe.

[0021] While the family of devices described in the aforementioned application are very useful in the analysis of a plurality of points within a target sample, there is a need to easily and automatically obtain such data on a full array of points so as to convert these data to an artificial image of the analytical findings over a large area of the sample. This is particularly important when heterogeneous samples, such as biological samples are examined with the non imaging volume microprobe. For instance, when examining tissues to determine the presence or absence of oncological pathologies, or other pathologies, visual techniques are followed, in some cases, by the resection of biopsy specimen. Such techniques are naturally limited in that the physician eye can only assess the visual appearance of potential pathologies, and the number of biopsies taken is by necessity limited. The appearance of pathological tissues does not provide information on the depth of the pathologies, and cannot provide positive diagnosis of the pathology. Furthermore, since biopsies are carried out ex vivo, a time lag between the taking of the biopsy and obtaining its results cannot be avoided. It would be very useful for physicians to have a device capable of performing such diagnostic tasks in vivo and to obtain differential diagnostics (between healthy and pathological tissues) while performing the examination. This is particularly important when performing exploratory surgical procedures, but can be very useful when examining more accessible tissues as well.

[0022] Where the diagnostic device is to come into contact with body tissues, there is a further need that its surfaces be insulated from contact with those tissues in order to avoid contamination. During sterile procedures, the device can introduce contamination into body tissues. Furthermore, the device can become contaminated by contact with the tissues of one patient and transmit that contamination to another patient. It is desirable that an apparatus that provides this insulation to the diagnostic device be compatible with the optical characteristics of the diagnostic device, so that the presence of the insulating apparatus does not impair the diagnostic device's accuracy or ease of use. It would be further desirable to provide an insulating apparatus that conforms to the anatomic area in which it is being used. For example, a differently shaped insulating apparatus may be required for diagnosing tissues through an endoscope than would be useful for diagnosing abnormalities of the cervix.

[0023] A number of devices have been described in the prior art relating particularly to confocal microscopy where illumination and detection arrays are provided. For instance, a confocal scanning microscope in which mechanical scanning of the illuminating and the transmitted (or the reflected) beams is avoided is described in U.S. Pat. No. 5,065,008. A light shutter array is used to provide synchronous detection of a scanned light beam without the need to move a photodetector to follow the scanning beam, and each of the shutters is serving, in essence, as a field stop in the confocal microscope. In other embodiments, two overlapping arrays of liquid crystals are used as optical shutter arrays to attempt reduction in the size of the field stops. As is well known in the art of confocal microscopy, in order to obtain the desired resolution afforded by this technique, the dimensions of the field stops need to be small relative to the diffraction limit of the optical beam used in the system. Other embodiments also provide for two sets of field stops, conjugated within the sample, one set for the illuminating beam and one set for the transmitted or reflected beam. While this patent teaches the use of electronic scanning of the illumination and response beams, the illumination intensity and response signal strength are drastically limited due to the use of dual liquid crystal optical shutters required to achieve the pin-hole effect of a scanning confocal microscope.

[0024] Another confocal imaging device is taught in U.S. Pat. No. 5,028,802, where a microlaser array provides a flying spot light source in a confocal configuration. Similarly U.S. Pat. No. 5,239,178 provides for an illuminating grid for essentially the same purpose, except that light emitting diodes are used for the grid's light sources. These approaches, however, are limited to monochromatic illumination and are usable only with relatively long wavelengths at which solid state laser diodes and thus microlaser arrays or light emitting diode arrays are available.

[0025] None of these devices provide for an array of non-imaging volume microprobes. Accordingly, there is a need for a device comprising an array of non-imaging volume microprobes in which a plurality of volume elements in a sample can rapidly be scanned in order to obtain diagnostic or analytical information over a relatively large area of the sample without integrating the data from all the sampled volume elements.

SUMMARY OF THE INVENTION

[0026] In the present invention, the principles taught in the aforementioned application are applied to automatically obtain optical responses from a three dimensional array of such volume elements by providing a plurality of non imaging volume microprobes in parallel which automatically presents mapping of the diagnostic information sought, in a plane generally parallel to the surface of the specimen (the xy plane) and in the z direction which is generally perpendicular to the xy plane.

[0027] The optical responses from an array of volume elements are further analyzed to provide visually (namely on a monitor) information which is not readily available by direct examination of the specimen. This is achieved by, in essence, providing an artificial three dimensional biochemical map composed from the optical responses, or more accurately, derivatives of such responses, of each individual volume element examined in an array, and by further converting these biochemical data to an artificial pathological image delineating the nature, extent and depth of pathologies observed. This is achieved by creating an artificial pathological scale, for each pathology of interest, by training the instrument to recognize specific pathologies. Specifically, a training set of specimens on which optical responses with a non imaging volume microprobe were collected, is subjected to a rigorous laboratory determination of the pathological state of each of its specimens and a value is assigned to each specimen on the artificial pathological scale. A set of linear equations relating to the responses (or functions of the responses) for each specimen to the pathological states, is constructed and optimized solutions for the correlation coefficients sought. These correlation coefficients are then used to transform responses obtained on unknown specimen to obtain the pathological state of these unknown specimen.

[0028] The objectives of the instant invention are achieved by providing an array of optical assemblies each consisting of two conjugated, or partially conjugated, optical assemblies. In each such assembly, the first optical assembly is designed to image selectively a transmitted beam from a light source, or another source of radiation, within a plurality of selected volume elements of a sample in a sequential manner. The second optical assembly is designed to collect light, or radiation emanating from the volume elements, in the same sequential manner, and transmit the collected light or radiation to a detector for further analysis of the interaction of the first transmitted beam with the volume elements. The first optical assembly includes a first field stop to achieve selective illumination of a selected volume element, and the second optical assembly includes a second field stop to restrict acceptance of said emanating radiation or light into the collection optics, essentially only from the selected volume element. Furthermore, a controller is provided to adjust the depth of the selected volume elements relative to the surface of the sample by controlling the respective focal points of the two optical assemblies while keeping them conjugated and having the volume element as a common conjugation point for both optical assemblies.

[0029] Sequential illumination of the various volume elements in an array is desired to assure that only responses from a given volume element are collected by the optical assembly associated with the volume element at any given time.

[0030] The sequential illumination of a plurality of volume elements can be carried out with a variety of devices. In some embodiments of the invention, an array of optical shutters is interposed between the light source and the sample, each shutter serving as either a field stop or an aperture stop for a specific optical assembly. In some embodiments, a single array of optical shutters is provided, while in other embodiments two arrays of optical shutters are provided. In yet another embodiment of the invention, an array of micromirrors is used to control the sequential illumination and response collection of the various volume elements in the sample. In yet another embodiment of the invention, an arrayed bundle of optical fibers is used to sequentially illuminate an array of volume elements in the sample and to collect sequentially responses from the volume elements. Appropriate movement of the optics so as to probe various depths of the sample is provided.

[0031] The optical responses from the selected volume elements bear important information about the volume elements, such as chemistry, morphology, and in general the physiological nature of the volume elements. When the sample is spectrally simple, these optical responses are analyzed by classical spectral techniques of peak matching, deconvolution or intensity determination at selected wavelengths. One such system could be the determination of the degree of homogeneity of a mixture or a solution of a plurality of compounds. However, when the samples are complex biological specimens, as mentioned above, the spectral complexity is often too great to obtain meaningful diagnosis. When such biological specimens are analyzed for subtle characteristics, we surprisingly found that the application of correlation transforms to spatially filtered optical responses obtained from an array of discrete volume elements, or the use of such transforms in conjunction with data obtained through non imaging microscopy, yields diagnostically meaningful results.

[0032] Specifically, we first select a training sample of a specific target pathology. Such a sample will preferably have at least 10 specimens. Optical responses are first collected from well defined volume elements in the specimens and recorded. These optical responses can be taken with an array microprobe or with a single volume microprobe device as described in the aforementioned co-pending application. The same volume elements that have been sampled with the non imaging volume microprobe are excised and biopsies (namely cytological analysis of the excised volume elements) is carried out in a classical pathological laboratory and the specimens are scored on an arbitrary scale which relates to the extent of the pathology, C (for instance a specific cancer) being characterized. These scores, C j , where C j is the score value assigned to the specimen j within the training set, should be as accurate as possible, and thus an average of a number of pathologists′ scores (determined on the same volume elements, j), can be used. We now create a set of equations Σa ic F(I ij )=C j , where i designates a relatively narrow spectral window (usually between 5 and 50 nm) and thus F(I ij ) is a specific function of the response intensity or other characteristics of the spectral response in the window i for volume element j. The function F is sometimes the response intensity itself, in that window, namely, F(I ij )=I ij , or F(I ij )=(dI ij )/dλ)/I ij , where λ is the median wavelength in the window i, or other functions. The factors a ic , the correlation transform's coefficients for the pathology C, are now found from the set of equations created above, by means well known in the prior art, such as multivariate linear regression analysis or univariant linear regression analysis. In such analysis, the number of wavelength windows i required to obtain faithful correlations between the optical responses and the pathological derivations of the values C j , is minimized and the set of correlation coefficients a ic for the pathology, C are found. When we now record the responses (I ik ) (which is a vector in the space of i optical windows, now minimized to a limited number of discrete elements) on a sample outside the training set and apply the transform operator (a ic ) on the vector F(I ik ), namely obtain the sum Σa ic F(I ik )=C k , we automatically obtain the score for the target pathology C for the volume element sampled.

[0033] It should be understood that other statistical tools, such as principal component regression analysis of the optical responses, could be used as well. One can also consider using in the correlation transforms, in lieu of functions of the optical responses at specific wavelengths, the Fourier transform of the total spectral responses. Furthermore, while taking the spectral responses from specific volume elements, these responses can be treated optically through either a spatial Fourier transform generator (such as a Sagnac interferometer) or a temporal Fourier transform generator (such as a Michelson interferometer), and then the data obtained can be used to create the desired correlation matrices to train the system for further data acquisition and image generation of the distribution of possible pathologies.

[0034] Instruments embodying the invention are deemed useful for obtaining artificial images of some characteristics of turbid materials, such as biological tissue, plastics, coatings, and chemical reaction processes, and may offer particular benefits in analysis of biological tissue, both in vitro and in vivo. To provide internal analysis, the invention is adapted to work with existing endoscopes, laparoscopes, or arthroscopes. To adapt the invention for diagnostic purposes involving contact with biological tissues, the invention can be provided with a covering that can be disposable to insulate the instrument from contact with biological tissues.

DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 is a schematic and generalized block diagram of the major elements of the present invention.

[0036] FIG. 2 is a block diagram of an embodiment of the invention with an array of light valves, in which each light valve acts as an addressable field stop for the illumination and detection beams.

[0037] FIG. 3 illustrates an embodiment where an array of lenslets of the same periodicity as the array of light valves acts as an array of objective lens for both the illumination and detection beams.

[0038] FIG. 4 and FIG. 4A illustrate embodiments of the invention in which separate illumination and detection light valves arrays create arrays of aperture stops, each in conjunction with lens arrays serving as objectives for the illumination and detection optics. In FIG. 4 A, the detection lens array is replaced with a single lens.

[0039] FIG. 5 illustrates an embodiment of the invention in which an array of (deformable) flat micromirrors is used as field stops and the sequential selection of micromirrors serves to sequentially illuminate volume elements in a sample.

[0040] FIGS. 6 and 6 A show embodiments of the invention in which an array of (deformable) off axis parabolic mirrors serve as selecting objectives to sequentially apply excitation beams to various volume elements and collect the responses from the volume elements.

[0041] FIG. 7 shows an embodiment of the invention in which the light shutter array is replaced with a fiber switching device to sequentially illuminate (and obtain responses from) an array of volume elements in a target sample.

[0042] FIG. 8 illustrates an embodiment having two optical assemblies, each coupled to its own (excitation and detection) fiber bundles in which sequential illumination of fibers (and detection) is practiced to obtain data from an array of volume elements.

[0043] FIGS. 9 and 10 illustrate embodiments of the invention in which light shutter arrays are coupled to optical fiber bundles.

[0044] FIG. 11 is a schematic representation of one of the embodiments of the invention, including a block diagram of the control and data processing elements of the system.

[0045] FIG. 12 is a block diagram that illustrates methods of using volume probe arrays of the invention, particularly in the diagnostic of various pathologies.

[0046] FIGS. 13 a and 13 b are bottom and top views, respectively, of a partial segment of a PVDF based optical shutter array.

[0047] FIG. 14 shows another embodiment of a PVDF based optical shutter array.

[0048] FIG. 15 is a top view of a micromachined optical shutter array.

[0049] FIG. 16 shows another embodiment of a micromachined optical shutter array.

[0050] FIG. 17 is an anatomic cross-sectional view showing an embodiment of the invention positioned within a body cavity within the female perineum.

[0051] FIG. 18 shows an embodiment of a disposable sheath illustrating optical fibers therein.

DETAILED DESCRIPTION OF THE INVENTION

[0052] In FIG. 1 we show a generalized schematic volume probe array, 10 , whose function is to collect data from a plurality of points in a target sample. The system generally includes an appropriate light source 11 , whose light output is conditioned and may be multiplexed in block 12 to create a plurality of light sources to be relayed to an array 13 of light valves. These light valves can act as illuminating field stops or aperture field stops, and only one valve is open at a given time, thus providing for sequential illumination of volume elements in sample 19 . The light emanating from each light valve is then directed to a targeted volume element in the sample 19 with an appropriate illumination objective 14 . In some embodiments, a single objective lens is used, while in other embodiments, we incorporate an array of objective microlens having the same periodicity as that of the light valve array.

[0053] Responses from each targeted volume element in the form of light emanating from the volume elements, is collected through a collection optics objective 15 (which in some embodiments can be the same as the illumination objective and an array of microlens), and through an array 16 of light valves (which can also be the same array as the one used for illumination). The responses are then directed to one or more detectors 17 to determine their optical and spectral characteristics.

[0054] It should be emphasized that both the illumination optics and the collection optics each contain a field stop having dimensions that are relatively large in relation to the average wavelength of the illuminating radiation, and furthermore, these field stops are conjugated to each other through the volume element examined. As a result, a well defined volume element is illuminated at any time, and the optical response from the element collected through the field stop of the collection optics is essentially limited to responses emanating from the volume element.

[0055] A controller 18 is provided to control the sequencing of the volume elements scanned (in the x,y plane, the plane of the sample) and to control the depth of the volume elements examined (in the z direction.)

[0056] In FIG. 2, a simple example of an array volume microprobe system 20 is shown. The system includes a light source 21 . Light from the light source is condensed with a lens 22 onto an array of light shutters 24 , through a beam splitter 25 . In this embodiment, each element 28 in the light shutter array serves as a field stop which is being imaged through an objective lens 26 on a sample 27 . The dimensions and shape of the shutters determine the morphology of volume elements sampled in a manner discussed in detail in copending applications, Ser. Nos. 08/510,041 and 08/510,043. In essence, the mean dimension, d, of each shutter is selected to be larger than the wavelength divided by the numerical aperture, NA, of the objective or d>λ/NA. Thus the image of the field stop in the plane of the sample is larger than the diffraction limited resolution for the wavelength. As a result, a very large proportion of the light that traverses a given field stop and is imaged in the sample is within a well-defined volume element of the sample. Similarly, while the total response to the illumination is distributed over a very large spatial angle (essentially 4π steradians), only responses that are emanating from within the same volume element are imaged back onto the field stop and reach detector 29 , by being reflected on the beam splitter 25 onto a collector lens 23 which concentrates the response onto the detector 29 . This results from the fact that the respective field stops of the illumination and detection systems are conjugated to each other via the target volume element. In the embodiment shown in FIG. 2 , both field stops are embodied within the same aperture (an optical shutter or a light valve 28 within the optical shutter array).

[0057] In some embodiments, the beam splitter 25 can be a dichroic mirror, particularly when the light source is a short wavelength (UV) exciting light source and the responses are fluorescence responses. In other embodiments, the beam splitter 25 can be a half silvered mirror which separates the optical path of responses from the sample from the optical path of the exciting beam, for instance, when the exciting beam is provided with a broad spectrum light source, and the responses involve back scattering and reflections from the sample (and thus mostly the extent of absorption of the exciting beam in the targeted volume element is examined).

[0058] The array of light valves, or optical shutters, can be implemented in a number of different ways. One can use liquid crystal sandwiched between two electrode arrays (deposited, as is in the prior art on transparent glass or plastic sheets in the form of transparent electrodes made of Indium Tin Oxide (ITO) or Tin Oxide (TO), usually doped with fluorine to provide good areal conductivity). One can also use films of PDLC (polymer dispersed liquid crystals) that might be easier to handle and have lower production costs. Another embodiment contemplated, when the required scanning is particularly fast, is an array of ferroelectric elements, each acting as a light valve. Yet another embodiment of the light valves can involve an array of PVDF (Polyvinyl-difluoride) bimorphs, each coated to be reflective (or opaque on both sides) on the side facing the light source and designed to bend out of the light path so as to create a light valve. The typical dimensions of the light valve range from a low of about 20 microns to as much as 1000 microns. The size is determined primarily by the application, the nature of the sample analyzed and the particular design of the specific array volume microprobe utilized. When using the general design of FIG. 2 , with a single large objective lens serving as a common objective to all the field stops in the array, the space between adjacent light valves is usually kept as small as possible, so as to provide as closely spaced as possible scanned volume elements. It should be understood however that in some embodiments, the spacing is kept relatively large (as large as the field stop itself) when an image of the pathology consisting of well spaced discrete points is more appropriate.

[0059] In operation, the controller 18 keeps one of the light valves open and adjusts the position of the device so as to image the field stop at the desired volume element in the sample 27 . Once the general position of the device relative to the sample has been optimized, the controller causes scanning of the surface of the specimen in the xy (the plane of the specimen) direction by sequentially closing an open light valve and opening an adjacent light valve. The time interval of each light valve in the open position is a strong function of the intensity of the light source and the efficiency of collection of the response from each volume element. In some embodiments, this time interval can be shorter than a millisecond, while in other embodiments tens to hundreds milliseconds are required.

[0060] The controller 18 also controls the position of the volume elements within the sample in the z direction, generally an axis perpendicular to the plane of the samples. This can be achieved in a number of ways. For instance, the whole optical assembly can be moved back and forth in the z direction. In some embodiments, this translational movement of the image plane of the field stops in the sample can be achieved by moving the objective alone, or the array of light valves, or both of these elements simultaneously. The specific design depends on the particular embodiment of the device.

[0061] It should be appreciated that since the intensity of illumination is highest within the volume element probed by the excitation beam (relative to zones surrounding the volume element), and that the response detected by the sensor 29 is primarily from the same volume element (and contains very little illumination emanating from zones surrounding the volume element), that changing the position of the volume element within the sample in the z direction will provide responses from various depths of the sample. This, in essence allows for analysis, in vivo of tissues at various depths, as long as the overall absorption of the illuminating beam by the tissue and the responses to the beam are not excessive.

[0062] In FIG. 3, a slightly different embodiment of an array volume microprobe 30 of the present invention is shown. The system includes a light source 31 with appropriate optics (not shown) to project a common field stop 41 through a condenser lens 32 onto an addressable shutter array 34 . Each element in the addressable shutter array can be considered an aperture stop which serves to further limit the spatial distribution of the light impinging on a sample 37 . In lieu of using a large singular objective (as used in the embodiment described in FIG. 2 ) to image the field stop on each volume element in the sample 37 , a lens array 36 is interposed between the shutter array and the sample. The lens array 36 consists of a plurality of microlens 42 . The periodicity of the lens array is exactly the same as that of the shutter array, and each lens 42 within the lens array 36 corresponds to a light valve 38 within the light shutter array 34 . In most embodiments, the light impinging on the shutter array would be collimated, and the shutter array would be fixed in position relative to the lens array. The volume element probed would be at the focal point of the objective lens within the microlens array, and movement of the combination of the shutter and lens array in the z direction, can be used to probe different layers within the sample, as explained above when describing the array volume microprobe of FIG. 2 . One can, however, conceive of other arrangements where the lens array and the shutter array are capable of moving independently, and probing the sample in the z direction is achieved by translation in the z direction of the lens array alone.

[0063] Light responses to the exciting radiation from the light source 31 from each of the sampled volume elements are collected through the same objective elements through which illumination is effected. The light responses are separated from the illuminating beam by the beam splitter 35 . These responses are then imaged via a collecting lens 33 onto a collection field stop 43 that restricts the responses received by a sensor 39 to be essentially only from the probed volume element. In operation, the controller 18 opens a given shutter and allows the illumination of a single volume element. Furthermore, the same light shutter allows optical responses to the excitation to be recorded by the detector 39 . This is followed by closing the light valve and opening another light valve, so that sequentially discrete volume elements within the sample are scanned to obtain optical responses thereof. One can scan all the desired volume elements in the array in a given x,y plane and then rescan the array at a different depth (in the z axis), so as to obtain three dimensional information on the target sample. One can also choose to operate the array volume microprobe in such a way that for each pixel, the respective light valve 38 is kept open, while the controller causes the shutter array together with the lens array to move in the z direction, thus probing at the same x,y location volume elements at various depths of the specimen.

[0064] In yet another embodiment of the array volume microprobe, the illuminating and detecting optics are each provided with their own array of optical shutters. In FIG. 4 , such an embodiment is shown schematically. Specifically, the array volume microprobe 50 includes a light source 51 , a first field stop 52 , a collimating lens 53 , a first shutter array 54 , a first objective lens array 55 , beam splitting means 56 , a second objective lens array 58 , a second shutter array 59 , a second collimating lens 60 , a second field stop 61 and a detector 62 . Not shown in FIG. 4 are appropriate means to image the light source 51 and detector 62 onto their respective field stops 52 and 61 . In operation, the light source 51 is imaged onto the field stop 52 , having dimensions that are greater than the diffraction resolution limits of the exciting radiation. The light emanating from the field stop 52 is collimated into an essentially parallel beam that impinges on the back side of the shutter array 54 . At any given time, only one of the light valves in the light shutter array is opened and its corresponding light valve in the detector shutter array is open. The sequential illumination of an array of volume elements within the sample, in a manner similar to that described above, coupled with the synchronous opening of the appropriate light valve in the detector array, assures that at any given time, only responses from the probed volume element are detected. Similarly, scanning in the x,y direction is provided by controller 18 sequencing the opening and closing of the light valves in the two shutter arrays in synchronism. It should be understood that in this embodiment, the two field stops 52 and 61 are conjugated to each other via each of the volume elements 63 in the sample 57 .

[0065] In FIG. 4 A, an arrangement essentially identical to that described in FIG. 4 is shown, except that the array of microlens 58 is replaced with a single large lens 58 ′. Like elements in FIGS. 4 and 4 A have the same reference numbers.

[0066] Yet another embodiment of the invention is illustrated in FIG. 5 , which shows an array volume microprobe 70 . The system includes a light source 71 and a detector 72 having their optical axes orthogonal to each other and separated by a first beam splitter 73 . The light emanating from the source is condensed onto an array of field stops 74 with a condenser lens 75 and a second beam splitter 76 . The array of field stops 74 consists of an array of micromirrors 77 that can be tilted in and out of a plane generally parallel to the plane of the array. Light reflected from any one of these mirrors, while in the untilted position, is reflected back through the second beam splitter 76 and is imaged onto a sample 79 with an objective lens 77 . As shown in FIG. 5 , only one micromirror 78 at any given time is oriented to reflect light onto the sample. All other micromirrors in the array are tilted so that light impinging on them is reflected away from the sample. In FIG. 5 , rays 1 and 4 are limiting rays for the total field, and rays 2 and 3 are limiting rays for a single micromirror. In operation, the micromirrors 77 are sequentially brought to the untilted position by the controller 18 , and as a result of this sequential untilting of micromirrors, a sequence of responses from volume elements in the sample 79 is recorded in the detector 72 . An artificial image from the responses can then be recorded and displayed. As in prior embodiments, probing of the sample with the volume microprobe array in the z direction (depth) can be achieved by either moving the objective lens 77 or the array 74 in the z direction.

[0067] The control of the tilting mirror is performed by controller 18 , and the tilting mechanism can be implemented in a number of ways well known in the prior art. For instance, the mirrors can be micromachined in silicon, leaving a cantilever in the middle of the back of the mirrors. Two opposing electrodes cause the mirror to tilt about the cantilever due to charging one or the other electrode with a charge opposing the charge on the mirror itself. Another method of obtaining tilting mirrors is well known in the art of deformable mirrors, whereby each micromirror is mounted on a bipolar piezoelectric element.

[0068] Yet another embodiment of the invention, a variation of the embodiment shown in FIG. 5 , is illustrated in FIG. 6 . An array volume microprobe 80 includes a light source 81 from which light is conditioned to pass through a first field stop (not shown) and through a lens 82 . The light is collimated onto an array of micromirrors 83 . The micromirrors are tiltable as described above. However, each of the micromirrors is shaped to be an off axis segment of a paraboloid of revolution having its focal point tracing an arc of radius which is somewhat larger than the distance of the array from the sample. The geometry is such that when the mirrors are untilted (parallel to the plane of the array), the axis of the paraboloid of revolution (of which the specific mirror is an off axis segment) is perpendicular to the plane of the array. Thus a line between the focal point (of the paraboloid of revolution) and the micromirror is at a predetermined angle to the normal to the array. The micromirrors can be tilted through that angle so as to bring the focal point of the paraboloid onto the sample.

[0069] In one embodiment of the invention, the micromirrors are arranged in alternating right rows 89 and left rows 88 of off-axis segments of a paraboloid. The right mirrors can be termed the exciting mirrors and the left mirrors the detecting mirrors. The focal points of each segment of right rows 89 , upon tilting at the above mentioned angle, resides within the sample at the volume element 85 , and its respective paraboloid axis of revolution is parallel to the optics axis of the exciting beam, while the tilting in the opposing direction (at the same angle) of each micromirror in an adjacent left row causes the focal point of each micromirror to move to the same volume element ( 85 ) in the sample 84 , and its respective paraboloid axis of revolution is parallel with the optics axis of the detector.

[0070] Thus, all mirrors in right rows 89 are used to excite volume element 85 in the sample 84 and all left rows 88 are used to collect responses from volume element 85 . In operation, only one pair of mirrors is tilted at any given time and the axes of all other mirrors point down toward the sample. As a result, an exciting beam from the light source 81 is imaged onto the volume element 85 , or more accurately, the first field stop is so imaged, while light impinging on all other mirrors is scattered away from the sample in all directions. Similarly, only responses emanating from the volume element 85 are imaged back onto the second field stop in front of the detector. As a result, a very high degree of discrimination is obtained, since the intensity of the exciting beam decreases very rapidly outside of volume element 85 , and responses from outside volume element 85 are essentially blocked by the second field stop in front of the detector 87 . The controller 18 controls the sequence of tilting each pair of mirrors to obtain an array of responses from different volume elements in the sample. The depth of the volume element in the sample is also controlled by the controller 18 by moving the total array 83 along the z axis toward or away from sample 84 .

[0071] A slightly modified embodiment of the volume probe array shown in FIG. 6 is presented in FIG. 6A . This embodiment allows for using each off axis parabolic micromirror both as an excitation and detection mirror. The system is equivalent to that shown in FIG. 6 and described above, except that the micromirrors of array 83 ′ of the volume probe array 80 ′ are rotated by 90° to the right or the left. Thus, in the unrotated position, the axis of revolution (and thus the optical axis) of each mirror is at 90° to the optical axis of the exciting and detecting optics. However, when a mirror 89 ′ is rotated by 90° to the right, its axis of revolution becomes parallel to the axis of the excitation, and the focal point of the off-axis parabolic micromirror is in volume element 85 ′. If an adjacent mirror 88 ′ is simultaneously rotated 90° to the left, then its axis of revolution becomes parallel to the detection optics, and its focal point is in volume element 85 ′. The volume element 85 ′ is determined by the overlap of the images of the two field stops, as explained in detail in copending application Ser. Nos. 08/510,041 and 08/510,043. In this embodiment, as well as the one shown in FIG. 6 , sheared conjugation of the excitation and detection optics field stops is used to provide for spatial discrimination of the excitation beam to the target volume element as well as the spatial discrimination of the detected responses to be essentially from each volume element. In operation, the controller 18 causes two adjacent micromirrors ( 89 ′ and 88 ′) to be rotated simultaneously as described above and thus provides excitation of essentially only the desired volume element 85 ′ and responses which emanate essentially from the volume element 85 ′.

[0072] An advantage of the embodiment shown in FIG. 6A is that a higher resolution of volume elements is feasible for the same density of micromirrors, since each mirror can be used to either excite a volume element or to collect responses from an adjacent volume element. This differs from the embodiment shown in FIG. 6 , where all left mirrors can be used only to collect responses and all right mirrors can be used only to excite volume elements. In the embodiment of FIG. 6 , the tilting of the off-axis paraboloids of each segment is in a plane perpendicular to the plane of the array, while in the embodiment of FIG. 6 A, the plane of rotation is parallel to the plane of the array.

[0073] In FIG. 7 , yet another embodiment of the invention is shown. An array microprobe 90 includes an illumination optical assembly with a light source 91 and a first collimating lens 92 , and a response collection optical assembly having a detector 93 and a second collimating lens 94 . The respective optical axes of the light source assembly and the detector assembly are at 90° to each other. A beam splitter 95 is positioned at the intersection of the exciting and detected beam so as to separate the detected signal from the excitation signal. A lens 96 is used to focus the exciting beam into an optical fiber 102 which is interfaced with a fiber switching element 97 . The fiber switching element 97 is terminated on the opposing side with a plurality of fibers 98 , and the switching element is capable of connecting optically and sequentially (under the control of the controller 18 ) the proximal fiber 102 to any of the fibers 98 in the distal bundle. The ends of the individual fibers in the bundle are then arranged in an array 99 (this array may be either a linear array or a two dimensional array). An objective lens 100 then images the respective ends of the fibers in the fiber bundle onto the specimen. Each individual fiber end (within the fiber holder) defines a field stop which is imaged onto the sample. This field stop serves as a field stop to both the exciting beam and the detected responses from the sample. As is described in more detail in copending application Ser. Nos. 08/510,041 and 08/510,043, such an arrangement involves the conjugation of both the exciting and detected optics via the volume elements in which the field stop is imaged, and thus provides for spatial discrimination of both the excitation beam and the responses to be essentially from each volume element associated with each fiber in the array.

[0074] In operation, the fiber switching element 97 directs the exciting beam sequentially through all the fibers in the bundle 98 . As a result, a plurality of volume elements in the sample 101 (having a distribution corresponding to the array of fibers in the bundle 98 ) are excited sequentially. Responses are collected through the same field stop (the natural aperture of each fiber end) and are separated from the exciting beam by the beam splitter 95 to be detected in detector 93 . In this manner, one obtains responses from an array of volume elements that can then be displayed as an artificial image of the sample. This embodiment has the advantage that a higher intensity of excitation is feasible, since the light source is used sequentially by the different fibers in the bundle.

[0075] In FIG. 8 , yet another embodiment of a volume microprobe array 110 is shown. The device includes two optical assemblies, an excitation or source assembly 111 and a detector assembly 112 . Each of the assemblies is interfaced to its own individual fiber bundle 113 and 114 , respectively. The individual fibers are organized, in one embodiment, in two rows 116 and 117 , respectively, in a fiber holder 115 . When a linear array is desired, the fibers are organized in two opposing rows, one row consisting of the excitation fibers in bundle 113 and the other row of detection fibers in bundle 114 . When a two-dimensional array is desired, the fibers are organized in alternating rows of excitation fibers and detection fibers, with a small tilt of such rows relative to each other. The excitation optics 111 includes a light source 118 and focusing optics 119 that can focus the output of the light source on each fiber in the bundle 113 sequentially. A rotating mirror 120 is used to index the light source onto the opening apertures of the fibers in the bundle 113 . One should appreciate that the input aperture of the excitation fibers can be terminated in an appropriate way to improve collection of the light from the rotating mirror. Such termination may include, but is not limited to, flaring of the input end of the fiber, or termination of each fiber with a small compound parabolic concentrator, as is well known in the prior art.

[0076] In operation, the controller 18 causes the incremental rotation of the mirror 120 so as to direct the excitation beam to fibers in bundle 113 sequentially. This light then emanates through an excitation field stop at each distal end of the fiber, the field stop being essentially the aperture of each fiber. These field stops are imaged onto a sample 124 with objective microlens at the end of each fiber. The distal ends of both excitation and detection fibers are terminated into microlenses that serve as objectives. The excitation and detection fibers may be at a slight angle to each other, and sheared conjugation of their respective field stops (fiber apertures) defines the volume elements probed. The volume elements in the sample will be a mirror image of the fiber arrangement, namely a row or an array of points, depending on the organization of the fibers in the fiber holder 115 . The distance between the volume elements may be the same as that between the fibers in the fiber holder or may differ from the interspacing of the fibers in the fiber holder, and will depend on the magnification of a relay lens 125 . In some embodiments, one can allow for movement of the relay lens relative to the fiber holder so as to provide magnification (or demagnification). However, the size of each volume element will also be modified somewhat.

[0077] This configuration allows for the sequential illumination of an array of volume elements in sample 124 . An excited volume element will emit a response to the exciting beam. To ensure that responses that are essentially emanating only from the desired volume element are detected, the responses are collected with a dedicated fiber from bundle 114 . The optics are configured such that the respective field stops of the response fibers (their natural aperture) are each conjugated to the respective the field stops of the associated exciting fiber. As a result of this conjugation (or, more accurately sheared conjugation, since the exciting and detecting field stops are slightly spaced apart), the excitation beam has its highest intensity within the sample within the zone of sheared conjugation (the probed volume element), and the intensity declines very rapidly outside the volume element. Furthermore, responses collected by each detection fiber emanate essentially only from the volume element, and any response collected from adjacent tissues is very small relative to the response obtained from the zone of sheared conjugation of the excitation and detection field stops.

[0078] Since the excitation of the array of volume elements is carried out sequentially, response will be transmitted through the fiber bundle 114 sequentially to the detecting optics 112 . In a preferred embodiment of the invention, the response optics include a receiving rotating mirror 123 which directs (sequentially and in synchronism with the excitation mirror 120 ) the responses through a focusing lens 122 to a detector 121 . This assures that stray responses (namely, responses emanating outside the zone of sheared conjugation and thus outside the target volume element) and collected by adjacent fibers, do not reach the detector. In this manner, as before, spatial discrimination is obtained, and sequential detection of responses from specific volume elements is achieved.

[0079] In this embodiment, the use of a beam splitter is avoided, and only very simple optics are used at the distal end of the device. Such a device is particularly suitable when a distance between the sample and the optics (source and detector) is required, such as in laparoscopic and endoscopic devices. This embodiment has the additional advantage that higher excitation energies are feasible, since the light source resources are not distributed simultaneously over a full array as in some embodiments described above, and in this respect is similar to the embodiment shown in FIG. 7 and described above.

[0080] In FIGS. 9 and 10 , two additional embodiments of the invention, which differ from each other only in the position in the system of the addressable light shutters, are shown. In FIG. 9, a volume microprobe array 130 that includes a light source 131 , a detector 132 , and an optical fiber bundle 133 is shown. The proximal end of the optical fiber bundle interfaces an addressable array of optical shutters 134 . The optical shutters are under the control of the controller 18 . Each fiber in the bundle 113 is positioned in an array arrangement that corresponds to the addressable light shutter array. The light source 131 is coupled to the light shutter array via a condenser lens 135 and a shutter array coupling lens 136 . As a result, light from the light source is distributed over the light shutter array, and, when one of the shutters is in the open position, light is transmitted to the specific fiber coupled to that specific shutter. At the distal end of the fiber bundle, the device has objective optics 137 which essentially images each of the apertures 138 of the fibers on a sample 139 . The distal apertures of the fibers are, in essence, acting as the excitation and detection field stops for each of the volume microprobes in the volume microprobe array 130 of this embodiment. Responses to the exciting signals emitted from volume elements in the sample are collected by the fibers through the same objective optics 137 and the same fibers through which excitation was carried.

[0081] Since the field stops of both the exciting and detecting optics are conjugated within the volume element probed, the excitations and responses are limited to the individual volume elements probed by each fiber. In operation, the light shutter array is controlled by the controller 18 to sequentially open the light shutters in front of the fiber bundle sequentially in such as way that only one fiber is powered at any given time. Thus, by the synchronous detection of responses from fibers that are coupled to an open shutter, a full artificial image of pathologies in the targeted sample can be constructed. As in some of the embodiments described above, the response is separated from the excitation by a beam splitter 140 positioned at 45° to the optical axis of the excitation optics and detection optics.

[0082] In FIG. 10, a similar volume microprobe array 150 is presented. The essential difference is that a shutter array 154 is positioned at the distal end of the fiber bundle. This allows for selecting an array of field stops determined by each of the apertures within the shutter array rather than by the individual apertures of the optical fibers.

[0083] In some embodiments of the volume microprobe arrays described above, a plurality of detectors corresponding to adjacent full regions of the shutter array are employed. Each detector accepts responses from a subarray of the light shutter array, and thus from the sample. In these embodiments, data collection is accelerated by the simultaneous opening of a light valve in each of the subarrays in the light shutter array and detecting the response in their respective detectors. When using this approach, care is taken to assure that interferences (or noise) from responses outside each specific region are smaller than a preset value of the expected response from the sample in each region.

[0084] In several embodiments described above, an array of light shutters is employed to sequence the excitation of an array of volume elements in the sample as well as collect responses from the volume elements. In some embodiments, each shutter serves as an excitation and detection field stop, while in other embodiments other optical elements in the system perform the function of the field stop. Such light shutters are well known in the prior art and have been used in a number of display devices, whereby the sequence of opening and closing sets of optical shutters that are back illuminated provide either a fixed or a time variable image.

[0085] The actual embodiments of such shutters in the prior art can take many forms. The ost widely used light shutter array is an array of liquid crystal elements having two sheets of polarizer one each on the front and the back of the array. On each element in the array, a voltage can be applied. When the voltage is sufficiently high, the liquid crystal causes rotation of the plane of polarization of light passing through it. The two polarizers are oriented in such a way that no light passes through an element when no voltage is applied. Thus, the polarizers are cross polarized (their relative orientation is 90°, thus the first polarizer removes all light polarized in one direction, while the second polarizer blocks the light passing through the then inactive liquid crystal element). When a sufficiently high voltage is applied, the plane of polarization of the light passing through the liquid crystal cell is rotated, so that the second polarizer is essentially transparent to the light passing through the active liquid crystal cell. The addressing can be carried out as in the prior art, either as row and columns, so that only the sum of voltages applied to both a row and column is sufficient to cause the desired rotation of polarized light. Since the dimensions of our light shutters are relatively large and the number of shutters small relative to the current practice in liquid crystal display, such addressing is quite sufficient, and cross talk is minimal and insignificant in view of the strong spatial discrimination due to the conjugation of the excitation and detection field stops.

[0086] When very large arrays are desired, approaches such as used in active matrix liquid crystals display (namely the activation of a pixel through the direct switching of an individual transistor at each pixel) can be practiced as well.

[0087] In yet another embodiment, the shutter array consists of ferroelectric elements activated in a manner similar to that of liquid crystal light shutter arrays. These shutter arrays are useful when the switching rate desired, namely, the rate of opening and closing a given light shutter in the array, is faster.

[0088] In yet another embodiment, the light switching medium is a polymer dispersed liquid crystal (PDLC). In such films, a dispersion of droplets of liquid crystal is embedded in a polymer having an index of refraction equal to the field oriented index of refraction of the liquid crystal dispersion. When no electrical field is applied, the droplets are randomly oriented and light is scattered in all directions. Thus the shutter can be considered as closed. When a sufficiently large electric field is applied to a PDLC element, the liquid crystal droplets orient themselves with the field and thus, in the direction of the field, the index of refraction is essentially constant and light passes through uninterrupted. Thus the shutter is open.

[0089] In yet another embodiment, essentially electromechanical shutters are used. Such can be easily implemented with piezoelectric bimorphs, which when actuated bend out of the path of the light and when inactive, assume a straight geometry which blocks light transmission through a given shutter.

[0090] In FIG. 13 a, a top view of a light shutter array <